Publications by authors named "Victor M Loyola-Vargas"

45 Publications

Tissue-specific proteome characterization of avocado seed during postharvest shelf life.

J Proteomics 2021 Mar 13;235:104112. Epub 2021 Jan 13.

Red de Estudios Moleculares Avanzados, Clúster Científico y Tecnológico BioMimic®, Instituto de Ecología A.C. (INECOL), Carretera Antigua a Coatepec No. 351, Congregación el Haya, CP 91070, Xalapa, Veracruz, Mexico. Electronic address:

Avocado is a nutritious and economically important fruit, generating significant income for exporter countries. Recently, by-products of this fruit such as seeds and peels, have raised interest in different industries. However, the biochemical features of the nutraceutical value of these tissues have not been analyzed using molecular approaches during the postharvest shelf life (PSL). We carried out comparative proteomics using tandem mass tagging (TMT) and synchronous-precursor selection (SPS)-MS. We analyzed testa, cotyledon, and embryo axes from avocado seeds at detachment from the tree (unripe), and after five (breaker) and ten days (ripe) of PSL. We identified 1968 proteins, from which 933 were specific to the testa, 167 to the embryo axis, and 23 to the cotyledon. The testa had a more dynamic proteome than the other tissues, resembling similar stress responses to those observed in peel tissues, such as down-accumulation of translational machinery, cell wall catabolism and synthesis of secondary metabolites. In contrast, the up-accumulation of the biosynthesis of l-glutamine, L-isoleucine, and l-serine was observed in all tissues. Our study provides the basic biochemical and physiological features of avocado seed during PSL and demonstrates that avocado seed tissues could potentially be used as a costless source of high-value compounds. SIGNIFICANCE: Avocado seed as a fruit by-product is a source of different valuable molecules, including those with nutraceutical properties. During PSL, several biochemical and physiological modifications occur in this dispersal unit, which also includes the alteration of several key metabolites' content. However, the proteome profile associated with different metabolic pathways that regulate the inner content of seed metabolites has not been previously studied. Our tissue-specific proteomics TMT-SPS-MS-based provides the first evidence of molecular and physiological changes in avocado tissues during PSL delivering fundamental knowledge of this organ. In this vein, the modulation of secondary metabolites, amino acid, and sugar metabolism of avocado tissues during PLS can encourage these by-products exploitation in multiple industries.
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http://dx.doi.org/10.1016/j.jprot.2021.104112DOI Listing
March 2021

Phenylpropanoids Are Connected to Cell Wall Fortification and Stress Tolerance in Avocado Somatic Embryogenesis.

Int J Mol Sci 2020 Aug 8;21(16). Epub 2020 Aug 8.

Red de Estudios Moleculares Avanzados, Instituto de Ecología A. C., Cluster BioMimic®, Carretera Antigua a Coatepec 351, Congregación el Haya, Xalapa, Veracruz CP 91073, Mexico.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including -coumaric acid and -ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.
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http://dx.doi.org/10.3390/ijms21165679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460882PMC
August 2020

YUCCA-Mediated Biosynthesis of the Auxin IAA Is Required during the Somatic Embryogenic Induction Process in .

Int J Mol Sci 2020 Jul 3;21(13). Epub 2020 Jul 3.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130 × 32 y 344 Col. Chuburná de Hidalgo, Mérida C.P. 97205, Mexico.

Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of (TAA) followed by the conversion of IPA to IAA by enzymes encoded by () genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in .
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http://dx.doi.org/10.3390/ijms21134751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369726PMC
July 2020

A Phosphoproteomic Analysis Pipeline for Peels of Tropical Fruits.

Methods Mol Biol 2020 ;2139:179-196

Red de Estudios Moleculares Avanzados, Clúster Científico y Tecnológico BioMimic®, Instituto de Ecología A.C. (INECOL), Veracruz, Mexico.

Phosphorylation is a posttranslational reversible modification related to signaling and regulatory mechanisms. Protein phosphorylation is linked to structural changes that modulate protein activity, interaction, or localization and therefore the cell signaling pathways. The use of techniques for phosphoprotein enrichment along with mass spectrometry has become a powerful tool for the characterization of signal transduction in model organisms. However, limited efforts have focused on the establishment of protocols for the analysis of the phosphoproteome in nonmodel organisms such as tropical fruits. This chapter describes a potential pipeline for sample preparation and enrichment of phosphorylated proteins/peptides before MS analysis of peels of some species of tropical fruits.
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http://dx.doi.org/10.1007/978-1-0716-0528-8_14DOI Listing
March 2021

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

The cell wall proteome from two strains of Pseudocercospora fijiensis with differences in virulence.

World J Microbiol Biotechnol 2019 Jul 2;35(7):105. Epub 2019 Jul 2.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A.C., Calle 43 No. 130 x 32 y 34, Col. Chuburná de Hidalgo, C.P. 97205, Mérida, Yucatán, Mexico.

Pseudocercospora fijiensis causes black Sigatoka disease, the most important threat to banana. The cell wall is crucial for fungal biological processes, including pathogenesis. Here, we performed cell wall proteomics analyses of two P. fijiensis strains, the highly virulent Oz2b, and the less virulent C1233 strains. Strains were starved from nitrogen to mimic the host environment. Interestingly, in vitro cultures of the C1233 strain grew faster than Oz2b in PDB medium, suggesting that C1233 survives outside the host better than the highly virulent Oz2b strain. Both strains were submitted to nitrogen starvation and the cell wall proteins were isolated and subjected to nano-HPLC-MS/MS. A total of 2686 proteins were obtained from which only 240 had a known function and thus, bioinformatics analyses were performed on this group. We found that 90 cell wall proteins were shared by both strains, 21 were unique for Oz2b and 39 for C1233. Shared proteins comprised 24 pathogenicity factors, including Avr4 and Ecp6, two effectors from P. fijiensis, while the unique proteins comprised 16 virulence factors in C1233 and 11 in Oz2b. The P. fijiensis cell wall proteome comprised canonical proteins, but thirty percent were atypical, a feature which in other phytopathogens has been interpreted as contamination. However, a comparison with the identities of atypical proteins in other reports suggests that the P. fijiensis proteins we detected were not contaminants. This is the first proteomics analysis of the P. fijiensis cell wall and our results expands the understanding of the fundamental biology of fungal phytopathogens and will help to decipher the molecular mechanisms of pathogenesis and virulence in P. fijiensis.
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http://dx.doi.org/10.1007/s11274-019-2681-2DOI Listing
July 2019

Signaling Overview of Plant Somatic Embryogenesis.

Front Plant Sci 2019 7;10:77. Epub 2019 Feb 7.

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

Somatic embryogenesis (SE) is a means by which plants can regenerate bipolar structures from a somatic cell. During the process of cell differentiation, the explant responds to endogenous stimuli, which trigger the induction of a signaling response and, consequently, modify the gene program of the cell. SE is probably the most studied plant regeneration model, but to date it is the least understood due to the unclear mechanisms that occur at a cellular level. In this review, the authors seek to emphasize the importance of signaling on plant SE, highlighting the interactions between the different plant growth regulators (PGR), mainly auxins, cytokinins (CKs), ethylene and abscisic acid (ABA), during the induction of SE. The role of signaling is examined from the start of cell differentiation through the early steps on the embryogenic pathway, as well as its relation to a plant's tolerance of different types of stress. Furthermore, the role of genes encoded to transcription factors (TFs) during the embryogenic process such as the , , and ) genes, Arabinogalactan-proteins (AGPs), and epigenetic factors is discussed.
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http://dx.doi.org/10.3389/fpls.2019.00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375091PMC
February 2019

Advanced Proteomic Approaches to Elucidate Somatic Embryogenesis.

Front Plant Sci 2018 20;9:1658. Epub 2018 Nov 20.

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

Somatic embryogenesis (SE) is a cell differentiation process by which a somatic cell changes its genetic program and develops into an embryonic cell. Investigating this process with various explant sources has allowed us to trace somatic embryo development from germination to plantlets and has led to the generation of new technologies, including genetic transformation, endangered species conservation, and synthetic seed production. A transcriptome data comparison from different stages of the developing somatic embryo has revealed a complex network controlling the somatic cell's fate, suggesting that an interconnected network acts at the protein level. Here, we discuss the current progress on SE using proteomic-based data, focusing on changing patterns of proteins during the establishment of the somatic embryo. Despite the advanced proteomic approaches available so far, deciphering how the somatic embryo is induced is still in its infancy. The new proteomics techniques that lead to the quantification of proteins with different abundances during the induction of SE are opening this area of study for the first time. These quantitative differences can elucidate the different pathways involved in SE induction. We envisage that the application of these proteomic technologies can be pivotal to identifying proteins critical to the process of SE, demonstrating the cellular localization, posttranslational modifications, and turnover protein events required to switch from a somatic cell to a somatic embryo cell and providing new insights into the molecular mechanisms underlying SE. This work will help to develop biotechnological strategies for mass production of quality crop material.
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http://dx.doi.org/10.3389/fpls.2018.01658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262180PMC
November 2018

A recent advance in the intracellular and extracellular redox post-translational modification of proteins in plants.

J Mol Recognit 2019 01 23;32(1):e2754. Epub 2018 Jul 23.

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

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant-pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S-nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S-nitrosylation on the structural conformation of well-known cell wall-associated proteins including pectin methylesterase and xyloglucan endo-transglycosylases.
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http://dx.doi.org/10.1002/jmr.2754DOI Listing
January 2019

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

Induction of Somatic Embryogenesis in Jatropha curcas.

Methods Mol Biol 2018 ;1815:207-214

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

Jatropha curcas has been a promising crop for biofuel production for the last decade. However, the lack of resistant materials to diseases and improved quality of the oil produced by the seeds has restricted the use of this promising crop. The genetic modifications in the fatty acid pathway, as well as the introduction of resistance to different diseases, would change the fate of Jatropha. To achieve these goals, we need to have a very efficient regeneration system. Here, we report a very useful protocol to induce somatic embryogenesis from leaves of Jatropha using cytokinin as the only growth regulator.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_13DOI Listing
March 2019

Auxin Immunolocalization in Coffea canephora Tissues.

Methods Mol Biol 2018 ;1815:179-188

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

Auxins are plant growth regulators that participate in a variety of biological mechanisms during the growth and development of plants. The most abundant natural auxin is indole-3-acetic acid (IAA). The physiological processes regulated by IAA depend on their temporal space accumulation in different tissues of a plant. This accumulation is regulated by its biosynthesis, conjugation, degradation, and transport. Therefore tools that allow us a qualitative and quantitative detection of IAA in plant tissues are very useful to understand the homeostasis of IAA during the life cycle of plants. In this protocol, the complete procedure for localization of IAA in different tissues of Coffea canephora is described using specific anti-IAA monoclonal antibodies.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_11DOI Listing
March 2019

Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.

Methods Mol Biol 2018 ;1815:131-148

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

Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_7DOI Listing
March 2019

An Introduction to Plant Tissue Culture: Advances and Perspectives.

Methods Mol Biol 2018 ;1815:3-13

Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico.

Plant tissue culture techniques are the most frequently used biotechnological tools for basic and applied purposes ranging from investigation on plant developmental processes, functional gene studies, commercial plant micropropagation, generation of transgenic plants with specific industrial and agronomical traits, plant breeding and crop improvement, virus elimination from infected materials to render high-quality healthy plant material, preservation and conservation of germplasm of vegetative propagated plant crops, and rescue of threatened or endangered plant species. Additionally, plant cell and organ cultures are of interest for the production of secondary metabolites of industrial and pharmaceutical interest. New technologies, such as the genome editing ones combined with tissue culture and Agrobacterium tumefaciens infection, are currently promising alternatives for the highly specific genetic manipulation of interesting agronomical or industrial traits in crop plants. Application of omics (genomics, transcriptomics, and proteomics) to plant tissue culture will certainly help to unravel complex developmental processes such as organogenesis and somatic embryogenesis, which will probably enable to improve the efficiency of regeneration protocols for recalcitrant species. Additionally, metabolomics applied to tissue culture will facilitate the extraction and characterization of complex mixtures of natural plant products of industrial interest. General and specific aspects and applications of plant tissue culture and the advances and perspectives are described in this edition.
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http://dx.doi.org/10.1007/978-1-4939-8594-4_1DOI Listing
March 2019

The role of chromatin modifications in somatic embryogenesis in plants.

Front Plant Sci 2015 18;6:635. Epub 2015 Aug 18.

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

Somatic embryogenesis (SE) is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques, and it is also an important technique to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene and gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate change in the genetic program of somatic cells, as well as moderating the transition between embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins and in vitro conditions modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the understanding of the role of epigenetic regulation of SE. In addition, we include a survey of different approaches to the study of SE, and new opportunities to focus SE studies.
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http://dx.doi.org/10.3389/fpls.2015.00635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539545PMC
September 2015

Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus.

PLoS One 2015 3;10(6):e0126414. Epub 2015 Jun 3.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, CP 97200, Mérida, Yucatán, México.

Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126414PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4454440PMC
February 2016

Biotic interactions in the rhizosphere: a diverse cooperative enterprise for plant productivity.

Plant Physiol 2014 Oct 12;166(2):701-19. Epub 2014 Aug 12.

Unidad de Biotecnología (C.D.) and Unidad de Bioquímica y Biología Molecular de Plantas (V.M.L.-V.), Centro de Investigación Científica de Yucatán, 97200 Merida, Yucatan, Mexico.

Microbes and plants have evolved biochemical mechanisms to communicate with each other. The molecules responsible for such communication are secreted during beneficial or harmful interactions. Hundreds of these molecules secreted into the rhizosphere have been identified, and their functions are being studied in order to understand the mechanisms of interaction and communication among the different members of the rhizosphere community. The importance of root and microbe secretion to the underground habitat in improving crop productivity is increasingly recognized, with the discovery and characterization of new secreting compounds found in the rhizosphere. Different omic approaches, such as genomics, transcriptomics, proteomics, and metabolomics, have expanded our understanding of the first signals between microbes and plants. In this review, we highlight the more recent discoveries related to molecules secreted into the rhizosphere and how they affect plant productivity, either negatively or positively. In addition, we include a survey of novel approaches to studying the rhizosphere and emerging opportunities to direct future studies.
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http://dx.doi.org/10.1104/pp.114.241810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213099PMC
October 2014

An efficient immunodetection method for histone modifications in plants.

Plant Methods 2013 Dec 16;9(1):47. Epub 2013 Dec 16.

Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Calle 43 No, 130, Col, Chuburná de Hidalgo, Mérida CP 97200, Yucatán, México.

Background: Epigenetic mechanisms can be highly dynamic, but the cross-talk among them and with the genome is still poorly understood. Many of these mechanisms work at different places in the cell and at different times of organism development. Covalent histone modifications are one of the most complex and studied epigenetic mechanisms involved in cellular reprogramming and development in plants. Therefore, the knowledge of the spatial distribution of histone methylation in different tissues is important to understand their behavior on specific cells.

Results: Based on the importance of epigenetic marks for biology, we present a simplified, inexpensive and efficient protocol for in situ immunolocalization on different tissues such as flowers, buds, callus, somatic embryo and meristematic tissue from several plants of agronomical and biological importance. Here, we fully describe all the steps to perform the localization of histone modifications. Using this method, we were able to visualize the distribution of H3K4me3 and H3K9me2 without loss of histological integrity of tissues from several plants, including Agave tequilana, Capsicum chinense, Coffea canephora and Cedrela odorata, as well as Arabidopsis thaliana.

Conclusions: There are many protocols to study chromatin modifications; however, most of them are expensive, difficult and require sophisticated equipment. Here, we provide an efficient protocol for in situ localization of histone methylation that dispenses with the use of expensive and sensitive enzymes. The present method can be used to investigate the cellular distribution and localization of a wide array of proteins, which could help to clarify the biological role that they play at specific times and places in different tissues of various plant species.
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http://dx.doi.org/10.1186/1746-4811-9-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868413PMC
December 2013

New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.

PLoS One 2013 20;8(8):e72160. Epub 2013 Aug 20.

Campus de Ciencias Exactas e Ingeniería, Universidad Autónoma de Yucatán, Mérida, Yucatán, México.

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY cotyledon1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-related homeobox4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748027PMC
January 2015

Appendix B: Plant biotechnology and tissue culture resources in the internet.

Methods Mol Biol 2012 ;877:419-25

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

This appendix compiles a list of useful Internet sites for cell culture scientists. A total of more than 100 sites have been selected, based on the quality of the information offered in them, as well as on their users' friendliness. We anticipate that some of these sites will be included among the reader's favorites (if they are not already).
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http://dx.doi.org/10.1007/978-1-61779-818-4_31DOI Listing
September 2012

Appendix A: The components of the culture media.

Methods Mol Biol 2012 ;877:407-17

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

The success in the technology and application of plant tissue culture is greatly influenced by the nature of the culture medium used. A better understanding of the nutritional requirements of cultured cells and tissues can help to choose the most appropriate culture medium for the explant used. It is also important to pay attention to a number of inaccuracies and errors which have appeared in several widely used plant tissue culture basal medium formulations.
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http://dx.doi.org/10.1007/978-1-61779-818-4_30DOI Listing
September 2012

Callus, suspension culture, and hairy roots. Induction, maintenance and characterization.

Methods Mol Biol 2012 ;877:29-40

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

The growth is a characteristic of each culture and it is determinate by the origin of the species, culture conditions, and type of culture. In this chapter, we make a comparison of the different growth parameters among three different species and three different types of cultures.
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http://dx.doi.org/10.1007/978-1-61779-818-4_3DOI Listing
September 2012

An introduction to plant cell culture: the future ahead.

Methods Mol Biol 2012 ;877:1-8

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

Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an experimental necessity for solving important fundamental questions in plant biology, but they currently represent very useful biotechnological tools for a series of important applications such as commercial micropropagation of different plant species, generation of disease-free plant materials, production of haploid and doublehaploid plants, induction of epigenetic or genetic variation for the isolation of variant plants, obtention of novel hybrid plants through the rescue of hybrid embryos or somatic cell fusion from intra- or intergeneric sources, conservation of valuable plant germplasm, and is the keystone for genetic engineering of plants to produce disease and pest resistant varieties, to engineer metabolic pathways with the aim of producing specific secondary metabolites or as an alternative for biopharming. Some other miscellaneous applications involve the utilization of in vitro cultures to test toxic compounds and the possibilities of removing them (bioremediation), interaction of root cultures with nematodes or mycorrhiza, or the use of shoot cultures to maintain plant viruses. With the increased worldwide demand for biofuels, it seems that PTC will certainly be fundamental for engineering different plants species in order to increase the diversity of biofuel options, lower the price marketing, and enhance the production efficiency. Several aspects and applications of PTC such as those mentioned above are the focus of this edition.
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http://dx.doi.org/10.1007/978-1-61779-818-4_1DOI Listing
September 2012

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

Plant Cell Physiol 2011 Aug 4;52(8):1401-21. Epub 2011 Jul 4.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, CP 97200, Mérida, Yucatán, México.

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.
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http://dx.doi.org/10.1093/pcp/pcr086DOI Listing
August 2011

Root secretion of phytochemicals in Arabidopsis is predominantly not influenced by diurnal rhythms.

Mol Plant 2010 May 12;3(3):491-8. Epub 2010 Feb 12.

Center for Rhizosphere Biology, Colorado State University, Fort Collins, CO 80523, USA.

Root-secreted phytochemicals mediate multiple interactions in the rhizosphere. The root exudation process can be altered by various biotic factors, including pathogenic and non-pathogenic microbes, and abiotic factors like temperature and soil moisture. It has been suggested that root secretion of specific flavonoids is influenced by diurnal rhythms (by light or dark) but a comprehensive analysis of the overall secretion of phytochemicals in response to diurnal rhythms has not been studied. In this study, we analyzed the effect of light/dark cycles on root exudation profiles using Arabidopsis as a model plant. Our results reveal that the root secretion of phytochemicals is partly regulated by the diurnal light cycle and follows two main patterns of secretion: (1) the large majority of phytochemicals in the exudates showed no diurnal pattern in their secretion, and (2) a few compounds showed a diurnal pattern in their secretion: three compounds increased in secretion only under light; two compounds increased in secretion only while it was dark; and two compounds increased in secretion during the transition from dark to light. Root-specific ABC transporters have been implicated in root exudation; an analysis of the gene expression patterns of ABC transporters in the roots of Arabidopsis at specific time points revealed that none of the ABC transporters followed a diurnal expression pattern, suggesting that they are expressed constantly during the day and night. Similarly, we analyzed the expression in roots of genes involved in secondary metabolite biosynthesis and found that some of the genes involved in phenylpropanoid and glucosinolate biosynthesis (i.e. 4-coumarate-CoA ligases (4CL1 and 4CL2), flavonol synthases (FS1 and FS2), and CYP79B3) followed distinct diurnal expression patterns. Overall, we have discovered that while root exudation of the majority of phytochemicals is constitutive, the secretion of a few compounds follows a diurnal rhythm, which is in accordance with the expression of some genes involved in secondary metabolism.
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http://dx.doi.org/10.1093/mp/ssq004DOI Listing
May 2010

Differential secretion and accumulation of terpene indole alkaloids in hairy roots of Catharanthus roseus treated with methyl jasmonate.

Mol Biotechnol 2009 Mar 8;41(3):278-85. Epub 2008 Oct 8.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, Merida, Yucatán, Mexico.

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.
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http://dx.doi.org/10.1007/s12033-008-9111-2DOI Listing
March 2009

Transcriptome analysis of Arabidopsis roots treated with signaling compounds: a focus on signal transduction, metabolic regulation and secretion.

New Phytol 2008 18;179(1):209-23. Epub 2008 Apr 18.

Center for Rhizosphere Biology, Colorado State University, Fort Collins, CO 80523, USA.

Gene expression in response to signaling molecules has been well studied in the leaves of the model plant species Arabidopsis thaliana. However, knowledge of gene expression and metabolic regulation at the root level is limited. Here, the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA) and nitric oxide (NO) were applied exogenously to induce various defense responses in roots, and their effect was studied using a combination of genomic, molecular and biochemical approaches. Genes involved in defense signaling/activation, cellular redox state, metabolism, transcription factors and membrane transport were altered in expression following treatment with SA, MeJA and NO. In addition, it was found that SA-, MeJA- and NO-elicited roots increased the root exudation of phytochemicals compared with the roots of nontreated control plants. Transport systems likely to be involved in the root exudation of phytochemicals, including the MATE, ABC, MFS, amino acid, sugar and inorganic solute transporters, showed altered expression profiles in response to treatments. Overall, significant differences were found in the signaling compound-elicited expression profiles of genes in roots vs those in leaves. These differences could be correlated to the underground nature of roots and their exposure to higher microbial inoculum rates under natural conditions.
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http://dx.doi.org/10.1111/j.1469-8137.2008.02458.xDOI Listing
September 2008

Possible role of light and polyamines in the onset of somatic embryogenesis of Coffea canephora.

Mol Biotechnol 2008 Jul 29;39(3):215-24. Epub 2008 Jan 29.

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 No. 130, Col. Chuburná de Hidalgo, CP 97200, Merida, Yucatan, Mexico.

The concentration of free and bound polyamines was studied during the somatic embryogenesis induction process in Coffea canephora explants. In the present study we show that when the induction of somatic embryogenesis in C. canephora is carried out under light conditions and in the presence of the plant growth regulator, benzylaminopurine, a cytokinin, a faster response to induction is obtained. In the darkness, the response is delayed for more than 20 days, and the number of embryos is smaller. In the absence of benzylaminopurine no embryogenic response was observed. The pronounced changes in the levels of putrescine, spermidine, and spermine, both free and bound, found in C. canephora suggest that a close correlation exists between polyamine biosynthesis and somatic embryogenesis in C. canephora during a period of cellular differentiation associated with the induction of somatic embryogenesis.
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http://dx.doi.org/10.1007/s12033-008-9037-8DOI Listing
July 2008

Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants.

Plant Physiol 2008 Feb 7;146(2):762-71. Epub 2007 Dec 7.

Center for Rhizosphere Biology, Colorado State University, Fort Collins, Colorado 80523, USA.

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.
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http://dx.doi.org/10.1104/pp.107.109587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245854PMC
February 2008

Membrane-associated phosphoinositides-specific phospholipase C forms from Catharanthus roseus transformed roots.

Mol Biotechnol 2007 Mar;35(3):297-309

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigaciòn Científica de Yucatán A.C., Calle 43 No. 130, Chuburná de Hidalgo, C. P. 97200, Mérida, Yucatán, México.

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.
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http://dx.doi.org/10.1007/BF02686015DOI Listing
March 2007