A hydrophobic proline-rich motif is involved in the intracellular targeting of temperature-induced lipocalin

Temperature-induced lipocalins (TILs) play an essential role in the response of plants to different abiotic stresses. In agreement with their proposed role in protecting membrane lipids, TILs have been reported to be associated to cell membranes. However, TILs show an overall hydrophilic character and do not contain any signal for membrane targeting nor hydrophobic sequences that could represent transmembrane domains. Arabidopsis TIL (AtTIL) is considered the ortholog of human ApoD, a protein known to associate to membranes through a short hydrophobic loop protruding from strands 5 and 6 of the lipocalin β-barrel. An equivalent loop (referred to as HPR motif) is also present between β-strands 5 and 6 of TILs. The HPR motif, which is highly conserved among TIL proteins, extends over as short stretch of eight amino acids and contains four invariant proline residues. Subcellular localization studies have shown that TILs are targeted to a variety of cell membranes and organelles. We have also found that the HPR motif is necessary and sufficient for the intracellular targeting of TILs. Modeling studies suggest that the HPR motif may directly anchor TILs to cell membranes, favoring in this way further contact with the polar group of membrane lipids. However, some particular features of the HPR motif open the possibility that targeting of TILs to cell membranes could be mediated by interaction with other proteins. The functional analysis of the HPR motif unveils the existence of novel mechanisms involved in the intracellular targeting of proteins in plants

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Abstract Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.This work was supported by grants of the Spanish Ministerio de Ciencia e Innovación (BIO2009-09523 to A.B., including European Regional Development Funds), the Spanish Consolider-Ingenio 2010 Program (CSD2007-00036 Centre for Research in Agrigenomics) and the Generalitat de Catalunya (2009SGR0026). DMA was recipient of a predoctoral fellowship from Generalitat de Catalunya (DEBEQ 2004).Peer Reviewe

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data

Identificació i caracterització de proteïnes relacionades en l'acumulació de carotenoides en el fruit del tomàquet

Els carotenoids són el major grup de pigments presents en la natura. Aquests compostos són molt apreciats al presentar la capacitat de prevenir malalties cardiovasculars, certs tipus de càncer i diversos processos degeneratius. Malgrat la necessitat d’aquests compostos els humans no són capaços de sintetitzar-los y s’han adquirir en la dieta. Essent el fruit del tomàquet la major font de carotenoids en la dieta occidental. El licopè i el β-carotè són els carotenoids que es produeixen en grans quantitats en el fruit del tomàquet al llarg del procés de maduració. L’acumulació de carotenoides provoca el característic color vermell del fruit i constitueix una de les principals característiques nutricionals del tomàquet madur. L’acumulació de licopè i β-caroteno se realitza dins d’un plast específic anomenat cromoplast. Actualment l’acumulació no ha estat extensivament estudiada, tot i que s’ha postulat que es realitza en entorns membranosos i mitjançat complexes lipoproteics (Vishnevetsky, et al. 1999). El coneixement dels mecanismes i proteïnes implicats en el procés és un dels passos claus per entendre l’acumulació i la informació pot ser utilitzada pera incrementar o modificar la quantitat de licopè i β-caroteno en el fruit, objectiu molt rellevant en els programes de millora del tomàquet. En aquest treball s’ha procedit a identificar i caracteritzar gens i proteïnes relacionades amb l’acumulació de carotenoids en el fruit de tomàquet des de dues aproximacions diferents. En la primera, es va determinar quins individus dins d’una població de línies de caràcter autofecunadatiu provinents del creuament dels tomàquet Solanum lycopersicum (Cv Money Maker) i Solanum pimpinellifollium presentaven nivells contrastants de carotenoids. Una cop detectades les línies contrastants, amb alts o baixos nivells de carotenoides, es van determinar quins eren els gens amb expressió diferencial en experiments de microarray, caracteritzant els més importants. I com a segona aproximació es va procedir a purificar, mitjançant tècniques proteòmiques, complexes lipoproteics i la posterior caracterització de las proteïnes detectades en ells.[spa] Los carotenoides son el mayor grupo de pigmentos presentes en la naturaleza. Estos compuestos son muy apreciados ya que presentan la capacidad de prevenir enfermedades cardiovasculares, ciertos tipos de cáncer y diversos procesos degenerativos. A pesar de la necesidad de estos compuestos los humanos no son capaces de sintetizarlos y los deben adquirir por la dieta. Siendo el fruto de tomate la mayor fuente de carotenoides en la dieta occidental. El licopeno y β-caroteno son los carotenoides que se producen en mayores cantidades en el fruto del tomate durante el proceso de maduración. La acumulación de dichos compuestos provoca el característico color rojo del fruto y constituye una de las principales características nutricionales del tomate maduro. La acumulación de licopeno y β-caroteno se realiza dentro de un plasto específico llamado cromoplasto. Actualmente la acumulación no ha sido extensivamente estudiada, aunque se ha postulado que se realiza en entornos membranosos y mediante complejos lipoproteicos (Vishnevetsky et al. 1999). El conocimiento de los mecanismos y proteínas implicados en el proceso es unos de los pasos claves para entender la acumulación y la información puede ser usada para incrementar o modificar la cantidad de licopeno y β-caroteno en el fruto, objetivo muy relevante en los programas de mejora del tomate. En este trabajo se ha procedido a identificar y caracterizar genes y proteínas relacionadas con la acumulación de carotenoides en el fruto de tomate desde dos aproximaciones diferentes. En la primera aproximación, se determino que individuos en una población de líneas de carácter autofecunadativo provinentes del cruce de Solanum lycopersicum (Cv Money Maker) y Solanum pimpinellifollium presentaban niveles contrastantes de carotenoides. Una vez detectadas las líneas contrastantes, con altos o bajos niveles de carotenoides, se determinaron cuales eran los genes de expresión diferencial mediante experimentos de microarray, llegando a caracterizar las más importantes. Y Como segunda aproximación se procedió a purificar, mediante técnicas proteómicas, complejos lipoprotéicos i la posterior caracterización de las proteínas detectadas en los mismos

A hydrophobic proline-rich motif is involved in the intracellular targeting of temperature-induced lipocalin

Temperature-induced lipocalins (TILs) play an essential role in the response of plants to different abiotic stresses. In agreement with their proposed role in protecting membrane lipids, TILs have been reported to be associated to cell membranes. However, TILs show an overall hydrophilic character and do not contain any signal for membrane targeting nor hydrophobic sequences that could represent transmembrane domains. Arabidopsis TIL (AtTIL) is considered the ortholog of human ApoD, a protein known to associate to membranes through a short hydrophobic loop protruding from strands 5 and 6 of the lipocalin β-barrel. An equivalent loop (referred to as HPR motif) is also present between β-strands 5 and 6 of TILs. The HPR motif, which is highly conserved among TIL proteins, extends over as short stretch of eight amino acids and contains four invariant proline residues. Subcellular localization studies have shown that TILs are targeted to a variety of cell membranes and organelles. We have also found that the HPR motif is necessary and sufficient for the intracellular targeting of TILs. Modeling studies suggest that the HPR motif may directly anchor TILs to cell membranes, favoring in this way further contact with the polar group of membrane lipids. However, some particular features of the HPR motif open the possibility that targeting of TILs to cell membranes could be mediated by interaction with other proteins. The functional analysis of the HPR motif unveils the existence of novel mechanisms involved in the intracellular targeting of proteins in plants

Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.Peer reviewe

Isolation of tomato fruit chromoplasts and determination of ATP levels

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.Peer reviewe

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data

Carotenoides en agroalimentación y salud

Los carotenoides son compuestos especiales; si bien es común referirse a ellos como pigmentos, lo cierto es que son compuestos de gran versatilidad e importancia en la naturaleza. Más específicamente, son de gran interés en agroalimentación y salud. Así, por ejemplo, son pigmentos naturales y por lo tanto tienen un importante papel en la elección de alimentos por parte de los consumidores. Asimismo, algunos de ellos son precursores de la vitamina A. Sin embargo, que cada vez exista más interés en los carotenoides en este contexto se debe en gran parte a muchos estudios de distinta naturaleza que indican que pueden proporcionar beneficios para la salud. Su interés en alimentación funcional es por lo tanto indudable. En este libro se refleja la experiencia en carotenoides de un gran número de profesionales de la región iberoamericana. En conjunto, se ofrece una visión general de la investigación sobre estos compuestos en agroalimentación y salud. Los autores son miembros de la red ibercarot (http://carotenoides.us.es), que tiene entre sus objetivos conformar una red estable y funcional de profesionales que aúnen esfuerzos en pos de identificar nuevas fuentes de carotenoides de interés nutricional, mejorar su producción y aumentar el valor de los productos que los contengan. Me gustaría agradecer a todas y cada una de las personas que han contribuido de una u otra forma a que este libro sea una realidad. Todos esperamos que sea de ayuda para personas interesadas en los temas desarrollados. Gracias especialmente al Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (Cyted, http:// www.cyted.org/) que, con su apoyo económico a la red ibercarot, ha hecho posible que varias decenas de equipos interaccionen en torno a temas de interés común para contribuir al desarrollo a distintos niveles de la región iberoamericana.RED TEMÁTICA IBERCAROT (referencia 112RT445) http://carotenoides.us.es PROGRAMA IBEROAMERICANO DE CIENCIA Y TECNOLOGIA PARA EL DESARROLLO – CYTEDPeer reviewe