Chloroplast iron transport proteins – Function and impact on plant physiology

12 Pags.- 1 Fig. 1 Tabl.Chloroplasts originated about three billion years ago by endosymbiosis of an ancestor of today’s cyanobacteria with a mitochondria-containing host cell. During evolution chloroplasts of higher plants established as the site for photosynthesis and thus became the basis for all life dependent on oxygen and carbohydrate supply. To fulfill this task, plastid organelles are loaded with the transition metals iron, copper, and manganese, which due to their redox properties are essential for photosynthetic electron transport. In consequence, chloroplasts for example represent the iron-richest system in plant cells. However, improvement of oxygenic photosynthesis in turn required adaptation of metal transport and homeostasis since metal-catalyzed generation of reactive oxygen species (ROS) causes oxidative damage. This is most acute in chloroplasts, where radicals and transition metals are side by side and ROS-production is a usual feature of photosynthetic electron transport. Thus, on the one hand when bound by proteins, chloroplast-intrinsic metals are a prerequisite for photoautotrophic life, but on the other hand become toxic when present in their highly reactive, radical generating, free ionic forms. In consequence, transport, storage and cofactor-assembly of metal ions in plastids have to be tightly controlled and are crucial throughout plant growth and development. In the recent years, proteins for iron transport have been isolated from chloroplast envelope membranes. Here, we discuss their putative functions and impact on cellular metal homeostasis as well as photosynthetic performance and plant metabolism. We further consider the potential of proteomic analyses to identify new players in the field.This work is supported by the Deutsche Forschungsgemeinschaft (DFG grantno.PH73/3–3toKP). DD was funded in the framework of theTransnational Cooperation (Germany, France, Spain) within the PLANT-KBBE Initiative funded by the Bundesministerium für Bildungund Forschung (BMBF grant no. FKZ:0315458A to KP, framework of the GABI initiative), and KP is funded by a Heisenberg fellowship of the DFG (grant no. PH73/6-1).Peer reviewe

Metal species involved in long distance metal transport in plants

20 Pags.- 2 Tabls.- 1 Fig. © 2014 Álvarez-Fernández, Díaz-Benito, Abadía, López-Millán and Abadía. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The mechanisms plants use to transport metals from roots to shoots are not completely understood. It has long been proposed that organic molecules participate in metal translocation within the plant. However, until recently the identity of the complexes involved in the long-distance transport of metals could only be inferred by using indirect methods, such as analyzing separately the concentrations of metals and putative ligands and then using in silico chemical speciation software to predict metal species. Molecular biology approaches also have provided a breadth of information about putative metal ligands and metal complexes occurring in plant fluids. The new advances in analytical techniques based on mass spectrometry and the increased use of synchrotron X-ray spectroscopy have allowed for the identification of some metal-ligand species in plant fluids such as the xylem and phloem saps. Also, some proteins present in plant fluids can bind metals and a few studies have explored this possibility. This study reviews the analytical challenges researchers have to face to understand long-distance metal transport in plants as well as the recent advances in the identification of the ligand and metal-ligand complexes in plant fluids.This study was supported by the Spanish Ministry of Economy and Competitiveness (projects AGL2010-16515 and AGL2012-31988), and the Aragón Government (group A03). Pablo Díaz-Benito was supported by a MINECO-FPI grant.Peer reviewe

Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply

<p>Abstract</p> <p>Background</p> <p>Plants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply.</p> <p>Results</p> <p>Root tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply.</p> <p>Conclusions</p> <p>The increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. Flavin synthesis could be involved in Fe uptake, whereas RFO sugars could be involved in the alleviation of oxidative stress, C trafficking or cell signalling. Our data also confirm the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways.</p

Estudio de la homeostasis de Fe y Mn en plantas mediante aproximaciones proteómicas

El Fe y el Mn están clasificados como micronutrientes que participan en funciones esenciales para el desarrollo y crecimiento de las plantas. El organismo no puede crecer sin un suministro adecuado de estos metales, pero por otro lado, si los niveles de exposición son demasiado elevados pueden resultar potencialmente tóxicos. El rango de concentraciones considerado fisiológico para los metales esenciales es muy estrecho y varía en función de la especie, el tejido y las condiciones de crecimiento. El conjunto de mecanismos implicados en el mantenimiento de los niveles adecuados de estos nutrientes es lo que se conoce como homeostasis. Desde un punto de vista fisiológico, la homeostasis de metales requiere la coordinación a nivel de toda la planta de los mecanismos de adquisición en la raíz, translocación a la parte aérea, almacenamiento y su posterior removilización a órganos sumidero. En la regulación de la homeostasis el sistema vascular juega un papel fundamental puesto que participa en la traslocación via xilema, en la removilización via floema y en la distribución y almacenamiento via apoplasto. Cuando este equilibrio se pierde, se producen pérdidas en la productividad causadas por disminuciones tanto en rendimiento como en la calidad de los cultivos que conllevan un alto impacto económico al generar productos con menor valor comercial y nutricional e incrementa los costes en el manejo del cultivo. La disponibilidad de ambos metales en la corteza terrestre depende del pH y las características redox del suelo, de forma que cuando el pH del suelo es elevado ambos metales se encuentran en formas con niveles de solubilidad mínimos que resultan difícilmente asimilables por las plantas. El 30% de los suelos cultivables presentan un pH elevado, indicando que la deficiencia de de estos metales resulta uno de los factores limitantes en el área mediterránea, en cultivos en el centro de Asia, el medio oeste de estados unidos o el sur de Australia. Por otro lado, en el caso del Mn, la cantidad del metal asimilable por las plantas aumenta considerablemente conforme el pH disminuye, de forma que en suelos con pH ácido puede llegar a resultar tóxico. Las actividades antropogénicas, como las actividades mineras y los residuos de incineración o de procesos industriales, han provocado el enriquecimiento en metales del medio ambiente, originándose una acumulación de 100 a 1.000 veces más alta en relación con su proporción natural en la corteza terrestre. La toxicidad de Mn supone un problema agronómico cada vez más común y es prevalentes en zonas del norte de Europa y Asia, el norte de EEUU y Canadá, gran parte de sudamérica o en algunas zonas de Australia. Aunque la información de la homeostasis en metales ha aumentado en los últimos años, todavía hay muchos interrogantes al respecto. Por ello, la proteómica puede resultar una herramienta muy útil a la hora de identificar y caracterizar nuevas proteínas que participan en la homeostasis de metales. El estrés por Fe ha sido más estudiado desde un punto de vista proteómico que el estrés por Mn. Los primeros estudios sobre Fe fueron en tejidos vegetales completos como la raíz y las hojas, y aún hoy en día los trabajos en estos tejidos son mayoritarios. En el caso del Mn, los trabajos publicados hasta el momento a nivel de proteoma se han centrado en plantas con características hiperacumuladoras o en estudios en hoja. Sin embargo, el análisis de subproteomas, entre los que se incluyen los fluidos de la planta, que resultan cruciales para el entendimiento de la homeostasis de estos metales, resultan todavía limitados. Con estos antecedentes el objetivo general de esta Tesis Doctoral ha sido incrementar el conocimiento existente acerca de los efectos que causan las deficiencias de Fe y Mn y la toxicidad por Mn en los proteomas vegetales implicados en la absorción (raíz) y el transporte (savias de xilema y floema y fluido apoplástico) de estos nutrientes mediante la aplicación de aproximaciones proteómicas. El uso de la proteómica diferencial en estos estreses y subproteomas puede proporcionar información sobre los procesos de adaptación de las plantas, así como apuntar hacia posibles estrategias para combatirlos. Además, el uso de técnicas de proteómica clásicas (2-DE) y avanzadas (“shotgun proteomics”) permite llevar a cabo la comparación entre técnicas y establecer las ventajas y limitaciones de ambas en el análisis de estos proteomas.<br /

Caracterización del proteoma de las envolturas interna y externa de cloroplasto de Pisum sativum

Comunicaciones a congreso

Root excretion and accumulation of riboflavin derivatives in iron-deficient Medicago truncatula

1 .pdf (43 Pags.) copia de la presentación original de los autores en el Simposio Internacional. Se adjunta también 1 .pdf con copia del "abstract" oficial.When grown in hydroponics under Fe deficiency, some Strategy I plant species develop yellow roots and cause a yellowing of the solution [1-2]. This phenomenon, first reported in the 60’s, is due to root accumulation and excretion of riboflavin and/or riboflavin derivatives such as riboflavin sulphates [3]. The function these compounds play in plant Fe efficiency is still not known, although roles in facilitating electron flow to the root Fe reductase and as antimicrobial agents in the rhizosphere have been hypothesized [4]. Any of these mechanisms may contribute to increase plant Fe efficiency. The aim of this work was to study flavin compounds present in roots of Fe-deficient Medicago truncatula. Plants were grown in Fe-sufficient nutrient solution (45 µM Fe) and in two Fe-deficient (0 µM Fe) nutrient solutions, either with CaCO3 (pH 8.0) or without CaCO3 (pH 5.5). Roots from Fe-sufficient plants were white and roots from Fe-deficient plants were yellow. Root morphology in the two Fe-deficient treatments was different, with swollen yellow tips at pH 8.0, and swollen tips (only some of them yellow) and yellow patches along their length at pH 5.5. A yellow colour was observed only in the Fe-deficient nutrient solution without CaCO3. Flavin compounds in the nutrient solution were concentrated in C18 Sep-Pack cartridges and eluted in methanol, and those in roots were extracted by grinding them with 100 mM ammonium acetate, pH 6.1. Flavin derivatives in root extracts and nutrient solution concentrates were separated by high performance liquid chromatography, and identification was carried out by ultraviolet-visible photodiode array spectrophotometry and electrospray ionization mass spectrometry, using time of flight (TOF) and quadrupole time of flight (QTOF) instruments. Root flavin accumulation and excretion depended on the plant Fe status and the presence of CaCO3 in the nutrient solution.In root extracts from Fe-sufficient plants only riboflavin was detected, whereas in roots of plants grown in both Fe deficiency treatments riboflavin and three different riboflavin derivatives were detected. Two of these derivatives were identified as 7α-hydroxyriboflavin and (E)-5-(4,5-dimethyl-2-((3R,4S)-2,3,4,5-tetrahydroxypentylamino)phenylimino)pyrimidine-2,4(3H,5H)-dione, the latter compound originated from the partial rupture of the riboflavin's isoalloxazine ring. In nutrient solutions, riboflavin and derivatives were detected only in Fe deficiency treatments, and the concentrations were much higher in nutrient solutions without CaCO3 than in those with CaCO3. As a conclusion, Fe-deficient M. truncatula roots accumulated and excreted riboflavin and three riboflavin derivatives different from those previously reported in plants. Further investigation is under way to identify the third flavin compound found.This study was supported by the Spanish Ministry of Science and Education (projects AGL2006-1416 and AGL2007-61948, co-financed with FEDER), the European Commission (EU 6th Framework Integrated Project ISAFRUIT), and the Aragón Government (group A03).Peer reviewe

Effects of Fe deficiency on the protein profiles and lignin composition of stem tissues from Medicago truncatula in absence or presence of calcium carbonate

12 Pags.- 2 Tabls.- 5 Figs.- Supp. Data.Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago truncatula. Two-dimensional electrophoresis separation of stem protein extracts resolved 276 consistent spots in the whole experiment. Iron deficiency in absence or presence of CaCO3 caused significant changes in relative abundance in 10 and 31 spots, respectively, and 80% of them were identified by mass spectrometry. Overall results indicate that Fe deficiency by itself has a mild effect on the stem proteome, whereas Fe deficiency in the presence of CaCO3 has a stronger impact and causes changes in a larger number of proteins, including increases in stress and protein metabolism related proteins not observed in the absence of CaCO3. Both treatments resulted in increases in cell wall related proteins, which were more intense in the presence of CaCO3. The increases induced by Fe-deficiency in the lignin per protein ratio and changes in the lignin monomer composition, assessed by pyrolysis-gas chromatography–mass spectrometry and microscopy, respectively, further support the existence of cell wall alterations. Biological significance: In spite of being essential for the delivery of nutrients to the shoots, our knowledge of stem responses to nutrient deficiencies is very limited. The present work applies 2-DE techniques to unravel the response of this understudied tissue to Fe deficiency. Proteomics data, complemented with mineral, lignin and microscopy analyses, indicate that stems respond to Fe deficiency by increasing stress and defense related proteins, probably in response of mineral and osmotic unbalances, and eliciting significant changes in cell wall composition. The changes observed are likely to ultimately affect solute transport and distribution to the leaves.Work supported by the Spanish Ministry of Science and Competitiveness (MINECO; projects AGL2012-31988, AGL2011-25379 and AGL2013-42175-R, co-financed by FEDER), the Aragón Government (group A03), and the US Department of Agriculture, Agricultural Research Service (under Agreement number 58-6250-0-008 to MAG). Support was obtained by contracts I3P-CSIC (JRC), FPI-MINECO (GL and LC-L), JAE-PRE-CSIC (EG-C) and JAE-DOC-CSIC (JR), co-financed by the European Social Fund.Peer reviewe

Çédille, revista de estudios franceses

Presentació

Adquisición y transporte de hierro en plantas

Las puntas de raíz de remolacha deficiente en Fe presentaron un engrosamiento subapical de color amarillo debido a la acumulación de flavinas, así como un aumento del número de pelos radiculares en estas zonas. La deficiencia de Fe indujo un aumento de 11 veces en la actividad reductasa férrica y de 4 veces en la capacidad de acidificación del medio, respuestas típicas de las raices de las plantas de estrategia I. La concentración de ácidos orgánicos y las actividades de diversos enzimas relacionados con la síntesis y metabolismo de estos compuestos aumentaron en las secciones amarillas de las puntas de raíz deficientes, indicando un aumento de la capacidad de fijar carbono, via fosfoenol piruvato carboxilasa, inducido por la deficiencia de Fe. Además, la deficiencia de Fe produjo un aumento en la concentración de nucleótidos de piridina, ATP, quinonas y en la tasa de consumo de oxígeno, surgiriendo la existencia de un aumento en la actividad mitocondrial en estas secciones amarillas, que proporcionaría poder reductor para la reductasa férrica. La concentración de ácidos orgánicos también aumentó en xilema y fluido apoplástico de hoja apoyando la existencia de un aumento en el flujo de C desde las raíces hasta las hojas de las plantas deficientes.Peer reviewe