Author: JOSE ANGEL HUERTA OCAMPO

Análisis proteómico de amaranto (Amaranthus hypochondriacus) bajo estrés por sequía

JOSE ANGEL HUERTA OCAMPO (2008)

"La sequía es uno de los principales factores que limitan la productividad agrícola mundial, las pérdidas originadas por sequía en cultivos de importancia económica son superiores a las provocadas por cualquier otro factor biótico o abiótico. La exposición de las plantas a ambientes con limitación de agua desencadena cambios fisiológicos regidos por la expresión de diferentes grupos de genes y proteínas cuya actividad ayuda a las plantas a tolerar condiciones ambientales adversas. Es necesario identificar los genes y proteínas que controlan la respuesta al estrés en cultivos de importancia económica para facilitar su mejoramiento biotecnológico a fin de obtener cultivos tolerantes y mejores rendimientos de las cosechas. El amaranto es un cultivo con gran potencial pues además de ser rico en proteínas y compuestos nutracéuticos, puede crecer en condiciones de poca agua y altas temperaturas, lo que lo convierte en un buen modelo para estudiar la tolerancia a sequía y estrés salino. El empleo de las herramientas moleculares ha contribuido al entendimiento de la percepción y transducción de señales en respuesta al estrés. El estudio de los mecanismos de respuesta al estrés en plantas mediante el enfoque proteómico es una herramienta más de la biología molecular que puede contribuir a identificar genes y rutas metabólicas que son cruciales para la tolerancia y la respuesta al estrés. En este trabajo se evaluó mediante electroforesis bidimensional (2-DE) los patrones de proteínas en hoja y raíz de plantas de amaranto sometidas a sequía. El análisis cuantitativo de los geles 2-DE permitió detectar incrementos y disminuciones en la concentración de proteínas tras el tratamiento aplicado, cuarenta y dos de estas proteínas se analizaron mediante espectrometría de masas. Lo cual mostró que las proteínas sobre-expresadas están relacionadas con la señalización en respuesta al estrés, eliminación de especies reactivas de oxígeno, síntesis de osmolitos, mantenimiento del correcto plegamiento y degradación de proteínas. Mientras que las proteínas que disminuyeron su concentración están relacionadas con daño al aparato fotosintético, disminución de la síntesis de lignina y disminución de la actividad de la glucólisis y el ciclo del ácido cítrico."

"Drought is one of the most important factors limiting the agricultural yield worldwide, losses by drought on important crops are higher than those occurred by any other biotic or abiotic stressing factor. Plant exposition to water limited environments leads physiological changes governed by the expression of different kinds of proteins and genes whose activity helps plants to tolerate adverse environmental conditions. It is necessary to identify the proteins controlling stress response on economical importance crops to facilitate its biotechnological improvement to obtain higher yields and better tolerant crops. Amaranth is a rediscovered crop with high potential, produces seeds with high content of proteins and is rich in nutraceutic compounds. It can grow at high temperatures with little quantity of water and is a good model for drought and salt stress studies. The usage of molecular tools has contributed to understand the perception and signal transduction in stress response. Proteomic analysis is a molecular biology tool that offers a new approach to discovering genes and molecular pathways related to stress responsiveness and tolerance. In this work, differential protein response in amaranth roots and leaves under severe drought stress was evaluated by two dimensional polyacrilamyde gel electrophoresis (2-DE). Quantitative analysis of the 2-DE gels spots made possible the identification of up and down-regulated proteins in response to severe drought. Forty two protein spots were analyzed by mass spectrometry. Identification of drought responsive proteins shows that up-regulated proteins are related with signaling, elimination of reactive oxygen species, osmolites synthesis and protein folding and degradation. Diminished responsive proteins to severe drought stress were related to photosynthetic damage, decrease in lignin synthesis and decrease in glucolysis and citric acid cycle activity."

Master thesis

Electroforesis bidimensional Estrés abiótico Amaranto BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR

Proteomic analysis of amaranth under abiotic stress

JOSE ANGEL HUERTA OCAMPO (2012)

"Commercial amaranth species such as Amaranthus hypochondriacus, A. cruentus and A. caudatus are native to America and are worldwide distributed. Amaranth is a highly nutritious and non-allergenic pseudo-cereal crop with remarkable nutraceutical properties. Amaranth is also a promising species often grown under semiarid conditions prone to both drought and salinity. For the above described amaranth is a good model to study tolerance to abiotic stress conditions such as drought and salt stress. Abiotic stress is one of the major factors limiting crop productivity worldwide and responsible for the reduction of up to 50% in the potential yield of crops. Plant exposure to abiotic stress triggers physiological changes ruled by the expression of different sets of genes and proteins whose activities enable plants to tolerate stressful environmental conditions. The application of 'omics' technologies has allowed the identification of genes and proteins related to abiotic stress tolerance. As part of the Amaranth-Future-Food project, in the present thesis, 2-DE gel coupled with LC-MS/MS approach was applied in order to analyze the changes in amaranth protein accumulation in response to abiotic stress conditions such as drought and salt stress. Some of the differentially accumulated proteins in response to stress may be considered associated to abiotic stress tolerance. However, functional characterization of these proteins in order to demonstrate its importance in plant response to abiotic stress is needed. Functional complementation by heterologous expression in yeast, gene silencing, heterologous expression in insertional lines and evaluation of differential accumulation of these proteins in hybrids from cultivars with contrasting tolerance to abiotic stress that preserve the desired trait are among the alternatives to demonstrate the role played by such proteins in plant responsive mechanisms to cope with abiotic stresses."

"Las especies comerciales de amaranto de grano Amaranthus hypochondriacus, A. cruentus y A. caudatus son nativas de América y están distribuidas en los cinco continentes. El amaranto es un pseudocereal altamente nutritivo y no alergénico con notables propiedades nutracéuticas, capaz de adaptarse a diversas condiciones de cultivo tales como suelos pobres en nutrientes y moderadamente salinos, condiciones de poca agua y altas temperaturas. Estas características lo convierten en un buen modelo para estudiar la tolerancia a condiciones de estrés abiótico como la sequía y el estrés salino. El estrés abiótico es uno de los principales factores que limitan la productividad agrícola mundial y genera la reducción de hasta el 50% en el rendimiento potencial de los cultivos. La exposición de las plantas a condiciones de estrés abiótico, desencadena cambios fisiológicos que se rigen por la expresión de diferentes grupos de genes y proteínas cuya actividad permite a las plantas tolerar condiciones ambientales adversas. El empleo cada vez más frecuente de las tecnologías “omicas” permite la identificación de genes y proteínas asociados con la tolerancia al estrés abiótico. Como parte del proyecto Amaranth-Future-Food, en este trabajo se evaluó mediante un enfoque proteómico la respuesta del amaranto a condiciones de estrés abiótico como la sequía y el estrés salino. Para lo cual se obtuvieron los patrones de proteínas mediante Electroforesis Bidimensional (2-DE) y se identificaron las proteínas diferencialmente acumuladas mediante Espectrometría de Masas en Tándem. Algunas de estas proteínas pueden considerarse como asociadas con la tolerancia al estrés abiótico. Sin embargo, es necesaria una caracterización funcional de estas proteínas a fin de demostrar su importancia en la respuesta de las plantas ante el estrés. Algunas alternativas para demostrar el papel que juegan estas proteínas diferencialmente acumuladas en la respuesta ante el estrés abiótico son: la complementación funcional de levaduras sensibles a componentes del estrés abiótico como el estrés osmótico, el silenciamiento génico, la expresión de dichas proteínas en líneas insercionales así como la evaluación de la acumulación diferencial de estas proteínas candidatas en híbridos que mantengan los rasgos deseados. Estos acercamientos nos darían una idea más clara del papel que desempeñan tales proteínas en los mecanismos de respuesta al estrés abiótico."

Doctoral thesis

BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR

Proteomic analysis of amaranth (Amaranthus hypochondriacus L.) leaves under drought stress

JOSE ANGEL HUERTA OCAMPO ERIKA PATRICIA BRIONES CERECERO ANTONIO DE LEON RODRIGUEZ ANA PAULINA BARBA DE LA ROSA (2009)

"Amaranth (Amaranthus hypochondriacus L.) is a plant that produces seed with high protein content, is rich on nutraceutical compounds, and can grow under environmental conditions where most of the basic crops are not able to develop. But little is know about the amaranth stress-responsive genes/proteins. The aim of this work was to apply the comparative proteomics approach to study the differential expression of amaranth leaf proteins under drought stress. However, the protein extraction from amaranth tissues is difficult as a result of high endogenous concentrations of interfering compounds; we have made some modifications of the classical trichloroacetic acid-acetone precipitation method to improve the quantity and quality of extracted proteins. Satisfactory and reproducible two-dimensional electrophoresis protein profiles were obtained; the method was also tested forAgave tequilana and Opuntia spp., two more examples of plants that are tolerant to drought stress. Drought-responsive proteins in amaranth leaves were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The upregulated proteins identified included chloroplast chaperonins involved in refolding and protein complexes protection. Downregulated proteins include Rubisco large subunit, cytochrome b6f, oxygen evolving complexes, and the ascorbate peroxidase mitochondrial. The results have shown that chloroplasts and mitochondria may play a central role in amaranth adaptation to abiotic stress, and further studies should be done at the subcellular level."

Article

Abiotic stress Agave LC/ESI‐MS/MS Nonmodel plants, Opuntia spp Recalcitrant plants BIOLOGÍA Y QUÍMICA

Low temperature conditioning of garlic (Allium sativum L.) "seed" cloves induces alterations in sprouts proteome

MIGUEL DAVID DUFOO HURTADO JOSE ANGEL HUERTA OCAMPO ALBERTO BARRERA PACHECO ANA PAULINA BARBA DE LA ROSA EDMUNDO MATEO MERCADO SILVA (2015)

"Low-temperature conditioning of garlic "seed" cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that "seed" bulbs from "Coreano" variety conditioned at 5 degrees C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic "seed" cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23 degrees C), and the other was conditioned at low temperature (5 degrees C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic "seed" cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies."

Article

Allium sativum Sprouts Cold conditioning Two-dimensional electrophoresis LC-ESI-MS/MS BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA

Proteomic analysis of non-toxic Jatropha curcas byproduct cake: fractionation and identification of the major components

ANDRES LEON VILLANUEVA JOSE ANGEL HUERTA OCAMPO ALBERTO BARRERA PACHECO SERGIO MEDINA GODOY ANA PAULINA BARBA DE LA ROSA (2018)

"Jatropha curcas non-toxic genotypes have been reported in Mexico and the press-cake, after oil extraction, represents a potential of new source of protein for food and feed uses. However, the characterization of the press-cake proteins is still unknown. The aim of this work was to carry out the molecular characterization of J. curcas seed storage proteins. Proteins in press-cake were pre-fractionated according to the classical Osborne procedure. Main protein fraction in J. curcas cake was represented by glutelins, the electrophoretic analysis showed that glutelins and globulins have the same profile, indicating that oil extraction process could have effect on globulins agglomeration. Protein fractions were analyzed by two-dimensional gel electrophoresis and mass spectrometry, results provide a new dataset of protein species or proteoforms that are accumulated in J. curcas endosperm. The identification of toxic proteins such as curcin in the non-toxic variety could represent that this protein have important roles in seeds. Regulatory proteins such as proteasome subunits and 14-3-3 were identified. A group of different heat shock and stress defense protein species was detected. Proteases related with inhibitory activity against DPPIV were also detected; this could support the potential use of J. curcas cake as nutraceutical food."

Article

Jatropha curcas LC–MS/MS Mass spectrometry Non-toxic Proteome Seed storage proteins BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR

The proteome map of the escamolera ant (Liometopum apiculatum Mayr) larvae reveals immunogenic proteins and several hexamerin proteoforms

José Ángel Huerta Ocampo María Soledad García Muñoz AIDA JIMENA VELARDE SALCEDO Eric Edmundo Hernández Domínguez JORGE LUIS GONZALEZ ESCOBAR Alberto Barrera Pacheco ALICIA GRAJALES LAGUNES Ana Paulina Barba de la Rosa (2018)

"The larvae of escamolera ant (Liometopum apiculatum Mayr) have been considered a delicacy since Pre-Hispanic times. The increased demand for this stew has led to massive collection of ant nests. Yet biological aspects of L. apiculatum larvae remain unknown, and mapping the proteome of this species is important for understanding its biological characteristics. Two-dimensional gel electrophoresis (2-DE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to characterize the larvae proteome profile. From 380 protein spots analyzed, 174 were identified by LC-MS/MS and homology search against the Hymenoptera subset of the NCBInr protein database using the Mascot search engine. Peptide de novo sequencing and homology-based alignment allowed the identification of 36 additional protein spots. Identified proteins were classified by cellular location, molecular function, and biological process according to the Gene Ontology annotation. Immunity- and defense-related proteins were identified including PPIases, FK506, PEBP, and chitinases. Several hexamerin proteoforms were identified and the cDNA of the most abundant protein detected in the 2-DE map was isolated and characterized. L. apiculatum hexamerin (LaHEX, GeneBank accession no. MH256667) contains an open reading frame of 2199?bp encoding a polypeptide of 733 amino acid residues with a calculated molecular mass of 82.41?kDa. LaHEX protein is more similar to HEX110 than HEX70 from Apis mellifera. Down-regulation of LaHEX was observed throughout ant development. This work represents the first proteome map as well as the first hexamerin characterized from L. apiculatum larvae."

Article

Escamoles Peptide de novo sequencing Gene ontology LC-MS/MS qRT-PCR Two-dimensional gel electrophoresis BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR