Scopoletin 8-Hydroxylase-Mediated Fraxetin Production is Crucial for Iron Mobilization

Iron (Fe) is an essential mineral nutrient and an important factor for the composition of natural plant communities. Low Fe availability in aerated soils with neutral or alkaline pH has led to the evolution of elaborate mechanisms that extract Fe from the soil solution. In Arabidopsis (Arabidopsis thaliana), Fe is acquired by an orchestrated strategy that comprises mobilization, chelation, and reduction of Fe3+ prior to its uptake. Here, we show that At3g12900, previously annotated as scopoletin 8-hydroxylase (S8H), participates in Fe acquisition by mediating the biosynthesis of fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin derived from the scopoletin pathway. S8H is highly induced in roots of Fe-deficient plants both at the transcript and protein levels. Mutants defective in the expression of S8H showed increased sensitivity to growth on pH 7.0 media supplemented with an immobile source of Fe and reduced secretion of fraxetin. Transgenic lines overexpressing S8H exhibited an opposite phenotype. Homozygous s8h mutants grown on media with immobilized Fe accumulated significantly more scopolin, the storage form of scopoletin, supporting the designated function of S8H in scopoletin hydroxylation. Fraxetin exhibited Fe-reducing properties in vitro with higher rates being observed at neutral relative to acidic pH. Supplementing the media containing immobile Fe with fraxetin partially rescued the s8h mutants. In natural Arabidopsis accessions differing in their performance on media containing immobilized Fe, the amount of secreted fraxetin was highly correlated with growth and Fe and chlorophyll content, indicating that fraxetin secretion is a decisive factor for calcicole-calcifuge behavior (i.e. the ability/inability to thrive on alkaline soils) of plants

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

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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

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

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

Proteómica diferencial: técnicas, aplicaciones y dificultades

El autor desarrolla su trabajo dentro del Grupo Fisiología del Estrés Abiótico en Plantas, Departamento de Nutrición Vegetal, Estación Experimental Aula Dei, EEAD (CSIC)Conocer el proteoma de un organismo es tener una imagen dinámica de todas las proteínas expresadas por ese organismo, en un momento dado y bajo determinadas condiciones concretas de tiempo y ambiente.Peer reviewe

Respuesta radicular a la deficiencia de Fe y la toxicidad por Cd

211 Pags. con Tabls. y Figs. Esta Tesis Doctoral es un compendio de trabajos previamente publicados y consta de las siguientes publicaciones: Andaluz, S., Rodríguez-Celma, J., Abadía, A., Abadía, J. y López-Millán, A.F. (2009). Time course induction of several key enzymes in Medicago truncatula roots in response to Fe deficiency. Plant Physiology and Biochemistry 47, 1082-1088. Rodríguez-Celma, J., Rellán-Álvarez, R., Abadía, A., Abadía, J. y López-Millán, A.F. (2010). Changes induced by two levels of cadmium toxicity in the 2-DE protein profile of tomato roots. Journal of Proteomics 73, 1694-1706. Rodríguez-Celma, J., Lattanzio, G., Grusak, M.A., Abadía, A., Abadía, J. y López-Millán, A.F. (2011). Root responses of Medicago truncatula plants grown in two different iron deficiency conditions: changes in root protein profile and riboflavin biosynthesis. Journal of Proteome Research 10, 2590-2601 Rodríguez-Celma, J., Vázquez-Reina, S., Orduna, J., Abadía, A., Abadía, J., Álvarez-Fernández, A. y López-Millán, A.F. (2011). Characterization of flavins in roots of Fe-deficient Strategy I plants, with a focus on Medicago truncatula. Plant & Cell Physiology, in press.El objetivo general de la tesis doctoral consiste en estudiar los efectos de la deficiencia de Fe y de la toxicidad por Cd en las raíces de dos plantas modelo, Solanum lycopersicum y Medicago truncatula, desde el punto de vista de la proteómica. Con este enfoque se pretende en primer lugar dibujar un mapa general de las alteraciones metabólicas producidas por ambos estreses nutricionales, y en segundo lugar utilizar esta plataforma para identificar posibles alteraciones desconocidas hasta la fecha que requieran un estudio en mayor profundidad. El estudio de estos estreses nutricionales tiene importancia desde el punto de vista agronómico, para evitar pérdidas de productividad, mejorar la calidad del cultivo y también para elaborar posibles estrategias de fitoremediación en suelos contaminados. Este proyecto también es interesante desde el punto de vista de seguridad alimentaria, con el fin último de establecer bases científicas para mejorar el contenido de Fe en cultivos comestibles o forrajeros y evitar el paso de Cd a la cadena trófica.Se plantean los siguientes objetivos específicos: 1- Caracterización de los cambios producidos por la toxicidad leve y severa por Cd en el perfil proteico de la raíz de tomate. 2- Caracterización de las respuestas fisiológicas de la raíz de Medicago truncatula a la deficiencia de Fe. 3- Caracterización de los cambios producidos por deficiencia de Fe directa e inducida por la presencia de carbonato cálcico en el medio de cultivo en el perfil proteico de la raíz de Medicago truncatula. 4- Identificación de los compuestos de naturaleza flavínica producidos en las raíces de Medicago truncatula deficientes en Fe y caracterización del patrón de acumulación y excreción de los mismos. 5- Estudio de la posible relación entre la inducción de la producción y excreción de compuestos de naturaleza flavínica y las respuestas principales de la Estrategia I en especies de interés hortícola.Peer reviewe