Publications by authors named "Elsa Góngora-Castillo"

22 Publications

  • Page 1 of 1

Eleven biosynthetic genes explain the majority of natural variation in carotenoid levels in maize grain.

Plant Cell 2020 Dec 2. Epub 2020 Dec 2.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.

Vitamin A deficiency remains prevalent in parts of Asia, Latin America, and sub-Saharan Africa where maize (Zea mays) is a food staple. Extensive natural variation exists for carotenoids in maize grain. Here, to understand its genetic basis, we conducted a joint linkage and genome-wide association study of the US maize nested association mapping panel. Eleven of the 44 detected quantitative trait loci (QTL) were resolved to individual genes. Six of these were correlated expression and effect QTL (ceeQTL), showing strong correlations between RNA-seq expression abundances and QTL allelic effect estimates across six stages of grain development. These six ceeQTL also had the largest percentage of phenotypic variance explained, and in major part comprised the three to five loci capturing the bulk of genetic variation for each trait. Most of these ceeQTL had strongly correlated QTL allelic effect estimates across multiple traits. These findings provide an in-depth genome-level understanding of the genetic and molecular control of carotenoids in plants. In addition, these findings provide a roadmap to accelerate breeding for provitamin A and other priority carotenoid traits in maize grain that should be readily extendable to other cereals.
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http://dx.doi.org/10.1093/plcell/koab032DOI Listing
December 2020

Transcriptomic analysis reveals key transcription factors associated to drought tolerance in a wild papaya (Carica papaya) genotype.

PLoS One 2021 29;16(1):e0245855. Epub 2021 Jan 29.

Centro de Investigación Científica de Yucatán A.C., Mérida, Yucatán, México.

Most of the commercial papaya genotypes show susceptibility to water deficit stress and require high volumes of irrigation water to yield properly. To tackle this problem, we have collected wild native genotypes of Carica papaya that have proved to show better physiological performance under water deficit stress than the commercial cultivar grown in Mexico. In the present study, plants from a wild Carica papaya genotype and a commercial genotype were subjected to water deficit stress (WDS), and their response was characterized in physiological and molecular terms. The physiological parameters measured (water potential, photosynthesis, Fv/Fm and electrolyte leakage) confirmed that the papaya wild genotype showed better physiological responses than the commercial one when exposed to WDS. Subsequently, RNA-Seq was performed for 4 cDNA libraries in both genotypes (susceptible and tolerant) under well-watered conditions, and when they were subjected to WDS for 14 days. Consistently, differential expression analysis revealed that after 14 days of WDS, the wild tolerant genotype had a higher number of up-regulated genes, and a higher number of transcription factors (TF) that were differentially expressed in response to WDS, than the commercial genotype. Thus, six TF genes (CpHSF, CpMYB, CpNAC, CpNFY-A, CpERF and CpWRKY) were selected for further qRT-PCR analysis as they were highly expressed in response to WDS in the wild papaya genotype. qRT-PCR results confirmed that the wild genotype had higher expression levels (REL) in all 6 TF genes than the commercial genotype. Our transcriptomic analysis should help to unravel candidate genes that may be useful in the development of new drought-tolerant cultivars of this important tropical crop.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245855PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845985PMC
January 2021

Improved method for isolation of high-quality total RNA from Weber roots.

3 Biotech 2021 Feb 15;11(2):75. Epub 2021 Jan 15.

CONACYT Research Fellow, Colegio de Postgraduados Campus Campeche, Carretera Haltunchen-Edzna km 17.5, Sihochac, 24450 Campeche, Mexico.

Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as and , as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.
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http://dx.doi.org/10.1007/s13205-020-02620-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810796PMC
February 2021

Seasonal shifts of arbuscular mycorrhizal fungi in Cocos nucifera roots in Yucatan, Mexico.

Mycorrhiza 2020 May 2;30(2-3):269-283. Epub 2020 Apr 2.

Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico.

The diversity and community structure of arbuscular mycorrhizal fungi (AMF) associated with coconut (Cocos nucifera) roots was evaluated by next generation sequencing (NGS) using partial sequences of the 18S rDNA gene and by spore isolation and morphological identification from rhizosphere soil. Root samples from six different Green Dwarf coconut plantations and from one organic plantation surrounded by tropical dry forest along the coastal sand dunes in Yucatan, Mexico, were collected during the rainy and dry seasons. In total, 14 root samples were sequenced with the Illumina MiSeq platform. Additionally, soil samples from the dry season were collected to identify AMF glomerospores. Based on a 95-97% similarity, a total of 36 virtual taxa (VT) belonging to nine genera were identified including one new genus-like clade. Glomus was the most abundant genus, both in number of VT and sequences. The comparison of dry and rainy season samples revealed differences in the richness and composition of AMF communities colonizing coconut roots. Our study shows that the main AMF genera associated with coconut tree roots in all samples were Glomus, Sclerocystis, Rhizophagus, Redeckera, and Diversispora. Based on glomerospore morphology, 22 morphospecies were recorded among which 14 were identified to species. Sclerocystis sinuosa, Sclerocystis rubiformis, Glomus microaggregatum, and Acaulospora scrobiculata were dominant in field rhizosphere samples. This is the first assessment of the composition of AMF communities colonizing coconut roots in rainy and dry seasons. It is of importance for selection of AMF species to investigate for their potential application in sustainable agriculture of coconut.
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http://dx.doi.org/10.1007/s00572-020-00944-0DOI Listing
May 2020

Data mining of metagenomes to find novel enzymes: a non-computationally intensive method.

3 Biotech 2020 Feb 30;10(2):78. Epub 2020 Jan 30.

3Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A. C., Mérida, Yucatán Mexico.

Currently, there is a need of non-computationally-intensive bioinformatics tools to cope with the increase of large datasets produced by Next Generation Sequencing technologies. We present a simple and robust bioinformatics pipeline to search for novel enzymes in metagenomic sequences. The strategy is based on pattern searching using as reference conserved motifs coded as regular expressions. As a case study, we applied this scheme to search for novel proteases S8A in a publicly available metagenome. Briefly, (1) the metagenome was assembled and translated into amino acids; (2) patterns were matched using regular expressions; (3) retrieved sequences were annotated; and (4) diversity analyses were conducted. Following this pipeline, we were able to identify nine sequences containing an S8 catalytic triad, starting from a metagenome containing 9,921,136 Illumina reads. Identity of these nine sequences was confirmed by BLASTp against databases at NCBI and MEROPS. Identities ranged from 62 to 89% to their respective nearest ortholog, which belonged to phyla Proteobacteria, Actinobacteria, Planctomycetes, Bacterioidetes, and Cyanobacteria, consistent with the most abundant phyla reported for this metagenome. All these results support the idea that they all are novel S8 sequences and strongly suggest that our methodology is robust and suitable to detect novel enzymes.
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http://dx.doi.org/10.1007/s13205-019-2044-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992798PMC
February 2020

Silencing against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem.

Plant Sci 2020 Feb 27;291:110360. Epub 2019 Nov 27.

Laboratori del Suro, Biology Department, Universitat de Girona, Campus Montilivi, E-17071, Girona, Catalonia, Spain. Electronic address:

Both suberin and its associated waxes contribute to the formation of apoplastic barriers that protect plants from the environment. Some transcription factors have emerged as regulators of the suberization process. The potato StNAC103 gene was reported as a repressor of suberin polyester and suberin-associated waxes deposition because its RNAi-mediated downregulation (StNAC103-RNAi) over-accumulated suberin and associated waxes in the tuber phellem concomitantly with the induction of representative biosynthetic genes. Here, to explore if other genes of the large NAC gene family participate to this repressive function, we extended the silencing to other NAC members by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c). Transcript profile of the StNAC103-RNAi-c phellem indicated that StNAC101 gene was an additional potential target. In comparison with StNAC103-RNAi, the silencing with StNAC103-RNAi-c construct resulted in a similar effect in suberin but yielded an increased load of associated waxes in tuber phellem, mainly alkanes and feruloyl esters. Globally, the chemical effects in both silenced lines are supported by the transcript accumulation profile of genes involved in the biosynthesis, transport and regulation of apoplastic lipids. In contrast, the genes of polyamine biosynthesis were downregulated. Altogether these results point out to StNAC101 as a candidate to repress the suberin-associated waxes.
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http://dx.doi.org/10.1016/j.plantsci.2019.110360DOI Listing
February 2020

Transcriptome analysis of the induction of somatic embryogenesis in and the participation of ARF and Aux/IAA genes.

PeerJ 2019 16;7:e7752. Epub 2019 Oct 16.

CONACYT Research Fellow-Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México.

Background: Somatic embryogenesis (SE) is a useful biotechnological tool to study the morpho-physiological, biochemical and molecular processes during the development of . Plant growth regulators (PGR) play a key role during cell differentiation in SE. The Auxin-response-factor (ARF) and Auxin/Indole-3-acetic acid (Aux/IAA) are fundamental components involved in the signaling of the IAA. The IAA signaling pathway activates or represses the expression of genes responsive to auxins during the embryogenic transition of the somatic cells. The growing development of new generation sequencing technologies (NGS), as well as bioinformatics tools, has allowed us to broaden the landscape of SE study of various plant species and identify the genes directly involved.

Methods: Analysis of transcriptome expression profiles of the genome and the identification of a particular set of differentially expressed genes (DEG) during SE are described in this study.

Results: A total of eight ARF and seven Aux/IAA differentially expressed genes were identified during the different stages of the SE induction process. The quantitative expression analysis showed that ARF18 and ARF5 genes are highly expressed after 21 days of the SE induction, while Aux/IAA7 and Aux/IAA12 genes are repressed.

Discussion: The results of this study allow a better understanding of the genes involved in the auxin signaling pathway as well as their expression profiles during the SE process.
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http://dx.doi.org/10.7717/peerj.7752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800528PMC
October 2019

DNA Methylation and Transcriptomic Next-Generation Technologies in Cereal Genomics.

Methods Mol Biol 2020 ;2072:65-84

CONACYT-Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico.

RNA sequencing (RNA-seq) coupled to DNA methylation strategies enables the detection and characterization of genes which expression levels might be mediated by DNA methylation. Here we describe a bioinformatics protocol to analyze gene expression levels using RNA-seq data that allow us to identify candidate genes to be tested by bisulfite assays. The candidate methylated genes are usually those that are low expressed in a particular condition or developmental stage.
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http://dx.doi.org/10.1007/978-1-4939-9865-4_7DOI Listing
December 2020

Elaboration of Transcriptome During the Induction of Somatic Embryogenesis.

Methods Mol Biol 2018 ;1815:411-427

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico.

Somatic embryogenesis (SE) is one of the most studied developmental processes due to its applications, such as plant micropropagation, transformation, and germplasm conservation. The use of massive techniques of sequencing, as well as the use of subtractive hybridization and macroarrays, has led to the identification of hundreds of genes involved in the SE process. These have been important developments to study the molecular aspects of the progress of SE. With the advent of the new massive techniques for sequencing RNA, it has been possible to see a more complete picture of whole processes. In this chapter we present a technique to handle the elaboration of the transcriptome from the extraction of RNA until the assembly of the complete transcriptome.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_29DOI Listing
March 2019

Novel Loci Underlie Natural Variation in Vitamin E Levels in Maize Grain.

Plant Cell 2017 Oct 2;29(10):2374-2392. Epub 2017 Oct 2.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Tocopherols, tocotrienols, and plastochromanols (collectively termed tocochromanols) are lipid-soluble antioxidants synthesized by all plants. Their dietary intake, primarily from seed oils, provides vitamin E and other health benefits. Tocochromanol biosynthesis has been dissected in the dicot , which has green, photosynthetic seeds, but our understanding of tocochromanol accumulation in major crops, whose seeds are nonphotosynthetic, remains limited. To understand the genetic control of tocochromanols in grain, we conducted a joint linkage and genome-wide association study in the 5000-line U.S. maize () nested association mapping panel. Fifty-two quantitative trait loci for individual and total tocochromanols were identified, and of the 14 resolved to individual genes, six encode novel activities affecting tocochromanols in plants. These include two chlorophyll biosynthetic enzymes that explain the majority of tocopherol variation, which was not predicted given that, like most major cereal crops, maize grain is nonphotosynthetic. This comprehensive assessment of natural variation in vitamin E levels in maize establishes the foundation for improving tocochromanol and vitamin E content in seeds of maize and other major cereal crops.
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http://dx.doi.org/10.1105/tpc.17.00475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774569PMC
October 2017

Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis.

Plant J 2016 12 27;88(5):775-793. Epub 2016 Sep 27.

The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA.

The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate-dependent decarboxylating enzyme that uses branched-chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched-chain amino acids), isotope labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate-dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.
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http://dx.doi.org/10.1111/tpj.13295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5195896PMC
December 2016

Transcriptome responses to Ralstonia solanacearum infection in the roots of the wild potato Solanum commersonii.

BMC Genomics 2015 Mar 26;16:246. Epub 2015 Mar 26.

Genetics Department, Universitat de Barcelona and Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB) Edifici CRAG, Campus UAB, Bellaterra, 08193, Catalonia, Spain.

Background: Solanum commersonii is a wild potato species that exhibits high tolerance to both biotic and abiotic stresses and has been used as a source of genes for introgression into cultivated potato. Among the interesting features of S. commersonii is resistance to the bacterial wilt caused by Ralstonia solanacearum, one of the most devastating bacterial diseases of crops.

Results: In this study, we used deep sequencing of S. commersonii RNA (RNA-seq) to analyze the below-ground plant transcriptional responses to R. solanacearum. While a majority of S. commersonii RNA-seq reads could be aligned to the Solanum tuberosum Group Phureja DM reference genome sequence, we identified 2,978 S. commersonii novel transcripts through assembly of unaligned S. commersonii RNA-seq reads. We also used RNA-seq to study gene expression in pathogen-challenged roots of S. commersonii accessions resistant (F118) and susceptible (F97) to the pathogen. Expression profiles obtained from read mapping to the S. tuberosum reference genome and the S. commersonii novel transcripts revealed a differential response to the pathogen in the two accessions, with 221 (F118) and 644 (F97) differentially expressed genes including S. commersonii novel transcripts in the resistant and susceptible genotypes. Interestingly, 22.6% of the F118 and 12.8% of the F97 differentially expressed genes had been previously identified as responsive to biotic stresses and half of those up-regulated in both accessions had been involved in plant pathogen responses. Finally, we compared two different methods to eliminate ribosomal RNA from the plant RNA samples in order to allow dual mapping of RNAseq reads to the host and pathogen genomes and provide insights on the advantages and limitations of each technique.

Conclusions: Our work catalogues the S. commersonii transcriptome and strengthens the notion that this species encodes specific genes that are differentially expressed to respond to bacterial wilt. In addition, a high proportion of S. commersonii-specific transcripts were altered by R. solanacearum only in F118 accession, while phythormone-related genes were highly induced in F97, suggesting a markedly different response to the pathogen in the two plant accessions studied.
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http://dx.doi.org/10.1186/s12864-015-1460-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4391584PMC
March 2015

A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.

Plant Cell 2014 Sep 16;26(9):3745-62. Epub 2014 Sep 16.

Department of Horticulture, Michigan State University, East Lansing, Michigan 48824

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.
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http://dx.doi.org/10.1105/tpc.114.130534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213168PMC
September 2014

Comparative transcriptome atlases reveal altered gene expression modules between two Cleomaceae C3 and C4 plant species.

Plant Cell 2014 Aug 8;26(8):3243-60. Epub 2014 Aug 8.

Institute of Plant Biochemistry, Cluster of Excellence on Plant Sciences, Heinrich-Heine-University, 40225 Düsseldorf, Germany

C(4) photosynthesis outperforms the ancestral C(3) state in a wide range of natural and agro-ecosystems by affording higher water-use and nitrogen-use efficiencies. It therefore represents a prime target for engineering novel, high-yielding crops by introducing the trait into C(3) backgrounds. However, the genetic architecture of C(4) photosynthesis remains largely unknown. To define the divergence in gene expression modules between C(3) and C(4) photosynthesis during leaf ontogeny, we generated comprehensive transcriptome atlases of two Cleomaceae species, Gynandropsis gynandra (C(4)) and Tarenaya hassleriana (C(3)), by RNA sequencing. Overall, the gene expression profiles appear remarkably similar between the C(3) and C(4) species. We found that known C(4) genes were recruited to photosynthesis from different expression domains in C(3), including typical housekeeping gene expression patterns in various tissues as well as individual heterotrophic tissues. Furthermore, we identified a structure-related module recruited from the C(3) root. Comparison of gene expression patterns with anatomy during leaf ontogeny provided insight into genetic features of Kranz anatomy. Altered expression of developmental factors and cell cycle genes is associated with a higher degree of endoreduplication in enlarged C(4) bundle sheath cells. A delay in mesophyll differentiation apparent both in the leaf anatomy and the transcriptome allows for extended vein formation in the C(4) leaf.
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http://dx.doi.org/10.1105/tpc.114.123752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371828PMC
August 2014

Architecture and evolution of a minute plant genome.

Nature 2013 Jun 12;498(7452):94-8. Epub 2013 May 12.

Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 36821 Irapuato, Guanajuato, México.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.
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http://dx.doi.org/10.1038/nature12132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972453PMC
June 2013

Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones.

Plant Cell Physiol 2013 May 15;54(5):686-96. Epub 2013 Mar 15.

Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chiba, 260-8675 Japan.

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.
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http://dx.doi.org/10.1093/pcp/pct040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653139PMC
May 2013

Bioinformatics challenges in de novo transcriptome assembly using short read sequences in the absence of a reference genome sequence.

Nat Prod Rep 2013 Apr;30(4):490-500

Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA.

Plant natural product research can be facilitated through genome and transcriptome sequencing approaches that generate informative sequence and expression datasets that enable characterization of biochemical pathways of interest. As the overwhelming majority of plant-derived natural products are derived from species with little, if any, sequence and/or genomic resources, the ability to perform whole genome shotgun sequencing and assembly has been and will continue to be transformative as access to a genome sequence provides molecular resources and a context for discovery and characterization of biosynthetic pathways. Due to the reduced size and complexity of the transcriptome relative to the genome, transcriptome sequencing provides a rapid, inexpensive approach to access gene sequences, gene expression abundances, and gene expression patterns in any species, including those that lack a reference genome sequence. To date, successful applications of RNA sequencing in conjunction with de novo transcriptome assembly has enabled identification of new genes in an array of biochemical pathways in plants. While sequencing technologies are well developed, challenges remain in the handling and analysis of transcriptome sequences. In this Highlight article, we provide an overview of the bioinformatics challenges associated with transcriptome analyses using short read sequences and how to address these issues in plant species that lack a reference genome.
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http://dx.doi.org/10.1039/c3np20099jDOI Listing
April 2013

Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

PLoS One 2012 26;7(12):e52506. Epub 2012 Dec 26.

Department of Plant Biology, Michigan State University, East Lansing, Michigan, United States of America.

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052506PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530497PMC
July 2013

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

J Biol Chem 2013 Feb 14;288(5):3163-73. Epub 2012 Dec 14.

Plant Biology Program, University of Kentucky, Lexington, Kentucky 40503, USA.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.
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http://dx.doi.org/10.1074/jbc.M112.415836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561538PMC
February 2013

Transcriptome analysis of symptomatic and recovered leaves of geminivirus-infected pepper (Capsicum annuum).

Virol J 2012 Nov 27;9:295. Epub 2012 Nov 27.

Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del I,P,N (Cinvestav)-Unidad Irapuato, Km 9,6 Libramiento Norte, Carretera Irapuato-León, Irapuato, Gto,, 36821, México.

Background: Geminiviruses are a large and important family of plant viruses that infect a wide range of crops throughout the world. The Begomovirus genus contains species that are transmitted by whiteflies and are distributed worldwide causing disease on an array of horticultural crops. Symptom remission, in which newly developed leaves of systemically infected plants exhibit a reduction in symptom severity (recovery), has been observed on pepper (Capsicum annuum) plants infected with Pepper golden mosaic virus (PepGMV). Previous studies have shown that transcriptional and post-transcriptional gene silencing mechanisms are involved in the reduction of viral nucleic acid concentration in recovered tissue. In this study, we employed deep transcriptome sequencing methods to assess transcriptional variation in healthy (mock), symptomatic, and recovered pepper leaves following PepGMV infection.

Results: Differential expression analyses of the pepper leaf transcriptome from symptomatic and recovered stages revealed a total of 309 differentially expressed genes between healthy (mock) and symptomatic or recovered tissues. Computational prediction of differential expression was validated using quantitative reverse-transcription PCR confirming the robustness of our bioinformatic methods. Within the set of differentially expressed genes associated with the recovery process were genes involved in defense responses including pathogenesis-related proteins, reactive oxygen species, systemic acquired resistance, jasmonic acid biosynthesis, and ethylene signaling. No major differences were found when compared the differentially expressed genes in symptomatic and recovered tissues. On the other hand, a set of genes with novel roles in defense responses was identified including genes involved in histone modification. This latter result suggested that post-transcriptional and transcriptional gene silencing may be one of the major mechanisms involved in the recovery process. Genes orthologous to the C. annuum proteins involved in the pepper-PepGMV recovery response were identified in both Solanum lycopersicum and Solanum tuberosum suggesting conservation of components of the viral recovery response in the Solanaceae.

Conclusion: These data provide a valuable source of information for improving our understanding of the underlying molecular mechanisms by which pepper leaves become symptomless following infection with geminiviruses. The identification of orthologs for the majority of genes differentially expressed in recovered tissues in two major solanaceous crop species provides the basis for future comparative analyses of the viral recovery process across related taxa.
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http://dx.doi.org/10.1186/1743-422X-9-295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546870PMC
November 2012

Genomic approaches for interrogating the biochemistry of medicinal plant species.

Methods Enzymol 2012 ;517:139-59

Department of Plant Biology, Michigan State University, East Lansing, Michigan, USA.

Development of next-generation sequencing, coupled with the advancement of computational methods, has allowed researchers to access the transcriptomes of recalcitrant genomes such as those of medicinal plant species. Through the sequencing of even a few cDNA libraries, a broad representation of the transcriptome of any medicinal plant species can be obtained, providing a robust resource for gene discovery and downstream biochemical pathway discovery. When coupled to estimation of expression abundances in specific tissues from a developmental series, biotic stress, abiotic stress, or elicitor challenge, informative coexpression and differential expression estimates on a whole transcriptome level can be obtained to identify candidates for function discovery.
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http://dx.doi.org/10.1016/B978-0-12-404634-4.00007-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926661PMC
March 2013

The capsicum transcriptome DB: a "hot" tool for genomic research.

Bioinformation 2012 6;8(1):43-7. Epub 2012 Jan 6.

Chili pepper (Capsicum annuum) is an economically important crop with no available public genome sequence. We describe a genomic resource to facilitate Capsicum annuum research. A collection of Expressed Sequence Tags (ESTs) derived from five C. annuum organs (root, stem, leaf, flower and fruit) were sequenced using the Sanger method and multiple leaf transcriptomes were deeply sampled using with GS-pyrosequencing. A hybrid assembly of 1,324,516 raw reads yielded 32,314 high quality contigs as validated by coverage and identity analysis with existing pepper sequences. Overall, 75.5% of the contigs had significant sequence similarity to entries in nucleic acid and protein databases; 23% of the sequences have not been previously reported for C. annuum and expand sequence resources for this species. A MySQL database and a user-friendly Web interface were constructed with search-tools that permit queries of the ESTs including sequence, functional annotation, Gene Ontology classification, metabolic pathways, and assembly information. The Capsicum Transcriptome DB is free available from http://www.bioingenios.ira.cinvestav.mx:81/Joomla/
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282275PMC
http://dx.doi.org/10.6026/97320630008043DOI Listing
August 2012