Accumulation and secretion of coumarinolignans and other coumarins in Arabidopsis thaliana roots in response to iron deficiency at high pH

22 Pags.- 3 Tabls.- 8 Figs. This Document is Protected by copyright and was first published by Frontiers (http://journal.frontiersin.org/journal/373). All rights reserved. it is reproduced with permission.Root secretion of coumarin-phenolic type compounds has been recently shown to be related to Arabidopsis thaliana tolerance to Fe deficiency at high pH. Previous studies revealed the identity of a few simple coumarins occurring in roots and exudates of Fe-deficient A. thaliana plants, and left open the possible existence of other unknown phenolics. We used HPLC-UV/VIS/ESI-MS(TOF), HPLC/ESI-MS(ion trap) and HPLC/ESI-MS(Q-TOF) to characterize (identify and quantify) phenolic-type compounds accumulated in roots or secreted into the nutrient solution of A. thaliana plants in response to Fe deficiency. Plants grown with or without Fe and using nutrient solutions buffered at pH 5.5 or 7.5 enabled to identify an array of phenolics. These include several coumarinolignans not previously reported in A. thaliana (cleomiscosins A, B, C, and D and the 5′-hydroxycleomiscosins A and/or B), as well as some coumarin precursors (ferulic acid and coniferyl and sinapyl aldehydes), and previously reported cathecol (fraxetin) and non-cathecol coumarins (scopoletin, isofraxidin and fraxinol), some of them in hexoside forms not previously characterized. The production and secretion of phenolics were more intense when the plant accessibility to Fe was diminished and the plant Fe status deteriorated, as it occurs when plants are grown in the absence of Fe at pH 7.5. Aglycones and hexosides of the four coumarins were abundant in roots, whereas only the aglycone forms could be quantified in the nutrient solution. A comprehensive quantification of coumarins, first carried out in this study, revealed that the catechol coumarin fraxetin was predominant in exudates (but not in roots) of Fe-deficient A. thaliana plants grown at pH 7.5. Also, fraxetin was able to mobilize efficiently Fe from a Fe(III)-oxide at pH 5.5 and pH 7.5. On the other hand, non-catechol coumarins were much less efficient in mobilizing Fe and were present in much lower concentrations, making unlikely that they could play a role in Fe mobilization. The structural features of the array of coumarin type-compounds produced suggest some can mobilize Fe from the soil and others can be more efficient as allelochemicals.Work supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (grant AGL2013-42175-R, co-financed with FEDER) and the Aragón Government (group A03). PS-T and AL-V were supported by MINECO-FPI contracts.Peer reviewe

Régulation de l'expression du gène de ferritine AtFer1 en réponse au fer chez Arabidopsis thaliana

Parmi les minéraux essentiels, le fer joue un rôle important dans de nombreux processus biologiques fondamentaux. De par sa réactivité avec l'oxygène, le fer est aussi toxique pour la cellule. Cette dualité du fer nécessite un strict contrôle de son homéostasie. Les ferritines sont des protéines ubiquitaires de stockage du fer sous forme non-toxique et remobilisable. L'objectif de ma thèse était d'identifier des éléments moléculaires impliqués dans la signalisation du statut nutritionnel en fer, en utilisant le gène AtFer1 d'Arabidopsis thaliana comme la cible terminale de cette voie de transduction. Chez les animaux, la synthèse des ferritines en réponse au fer est contrôlée au niveau post-transcriptionnel par le système IRE/IRP où IRP1 est une aconitase cytosolique. Chez Arabidopsis, nous avons identifié des trois homologues à IRP1 (ACO1 à -3) et par des approches de génétique inverse nous avons montré que les aconitases végétales ne sont pas impliquées dans la régulation de la synthèse des ferritines en réponse au fer. Chez les végétaux, la synthèse des ferritines est principalement régulée au niveau transcriptionnel par un excès de fer par l'intermédiaire d'une séquence cis-régulatrice particulière, l'IDRS. Par des approches pharmacologiques couplées à des analyses de microscopie nous avons identifié plusieurs éléments impliqués dans cette voie de signalisation. Le fer provoque une production de NO dans les plastes des cellules d'Arabidopsis. En aval de cette production de NO, une phosphatase de type PP2A est un régulateur positif dans la voie de signalisation. Un répresseur, actif en absence de fer, est dégradé par la voie dépendante de l'ubiquitine et du protéasome 26S en présence de fer et un facteur de transcription est fixé sur la séquence IDRS indépendamment du statut nutritionnel en fer. Ces approches nous ont également conduit à la mise en évidence d'un autre mécanisme de régulation de l'expression d'AtFer1 au niveau post-transcriptionnel. Ce mécanisme régule négativement l'accumulation des transcrits en réponse au fer en contrôlant la stabilité des transcrits AtFer1. Deux éléments (séquences DST et/ou ARN antisens) sont potentiellement impliqués dans cette dégradation des transcrits AtFer1. L'intégration de ces différents niveaux de régulation permet de contrôler finement la synthèse des ferritines en réponse au fer. Ce travail a permis d'appréhender les mécanismes de signalisation du statut en fer des plantes aboutissant in fine à la régulation de programmes génétiques permettant l'adaptation à des conditions environnementales fluctantesAmong essential mineral element, iron plays an important role in many biological processes. However, iron physicochemical properties leads to cellular toxicity. Iron homeostasis needs to be tightly controlled. Among the mechanisms involved in iron homeostasis, ferritins are of major importance. Ferritins are ubiquitous multimeric proteins able to store iron in a soluble and non-toxic form. My work aims at identifying molecular elements involved in sensing and signaling of iron nutrient status in plant cells by using the promoter of the ferritin encoding gene AtFer1 as the terminal target of this transduction pathway. In animals, ferritin synthesis is controlled by iron at post-transcriptional level via IRE/IRP binding where IRP1 is a cytosolic aconitase. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1 to 3. By reverse genetic approaches, Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. Indeed, in plants, ferritin synthesis is induced by iron excess, mainly at transcriptional level. A cis regulatory sequence (IDRS) is involved in this mechanism. By combining pharmacological and imaging approaches in an Arabidopsis cell culture system, we have identified several elements in the signal transduction pathway leading to the increase of AtFer1 transcript level after iron treatment. Nitric oxide quickly accumulates in the plastids after iron treatment. This compound acts downstream of iron and upstream of a PP2A-type phosphatase to promote an increase of AtFer1 mRNA level. A repressor acts in low iron condition and is ubiquitinated upon iron treatment and subsequently degraded through a 26 S proteasome-dependent pathway. A nuclear factor, different from the repressor, is able to bind the IDRS independently of iron status. These approaches allow us to discover another regulation mechanism occuring at the post-transcriptional level. Surprisingly, in Arabidopsis cells, iron treatment leads to rapid destabilization of AtFer1 mRNA. The increase of the degradation rate impacts strongly the half-life of ferritin transcripts. Two putative elements (DST sequences and/or antisense RNA) could be involved in this degradation mechanism of AtFer1 mRNA. This new post-transcriptional regulatory mechanism seems to be involved in the tightly control of ferritin expression in response to environmental variations. This work should contribute to understand molecular events involved in iron homeostasis in plant, therefore controlling the plant adaptation to fluctuation of environmental conditionsMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Keeping nitrate in the roots: an unexpected requirement for cadmium tolerance in plants.

International audienceLi et al. (2010) identified AtNRT1.8 as a membrane transporter involved in the control of long-distance transport of nitrate between roots and shoot. Both the regulation of AtNRT1.8 expression and the phenotype of an nrt1.8 mutant indicate that this transporter plays an important role in protecting the plant against cadmium toxicity, and possibly against a wide range of biotic and abiotic stresses

Les Ferritines chez A. thaliana (fonction et régulation)

Fe is essential for all cells because it is the cofactor of numerous proteins, however, excess free Fe is potentially deleterious for the cell. Ferritins are multimeric proteins, present in all the kingdoms of life that can store iron in a safe and bioavailable form. In mammals, ferritins are the main Fe store. They have been predicted to fulfil the same function in plants, but direct evidences are lacking. In plants, ferritin synthesis in response to iron overload is mainly regulated at the transcriptional level, whereas, in animals, it is mainly regulated at the post-transcriptional level by the aconitase dependent IRP/IRE system. The aims of my PhD project were: (i) to elucidate ferritin function in plant physiology and (ii) to decipher the signaling pathway leading to ferritin accumulation in response to iron overload. (i) To directly study ferritin function in plants, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. The loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporter genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, I showed that in the absence of ferritin, plants had higher levels of ROS, and increased activity of enzymes involved in their detoxification. Ferritins are also involved in iron-detoxification during senescence to avoid ROS accumulation. Seeds ferritins are also involved in the protection against oxidative stress during germination and appear to take part in the integrated iron homeostasis establishment. Taken together, my work showed that Arabidopsis ferritins are essential factors that integrate iron and redox homeostasis, while they do not constitute a major iron source for development. (ii) To study ferritin regulation in A. thaliana, the characterization of mutants in the three genes encoding aconitase permitted us to demonstrate that the IRP/IRE system does not occur in the regulation of iron metabolism in plants. Nevertheless, AtFer1 mRNA stability studies have revealed that iron treatment leads to the destabilization of the AtFer1 mRNA. We identified the presence of DST sequences, characterized as mRNA stability determinant, in the 3'-UTR of AtFer1 mRNA. Using chimeric constructs in which the AtFer1 3'-UTR or the AtFer1 3'-UTR with a mutated DST sequence were fused downstream of reporter genes, we have shown that the DST sequence in the 3'-UTR of AtFer1 is functional and sufficient for the iron-dependent mRNA degradation. Using dst1 and dst2 mutants, which are unable to destabilize transcript via DST sequences, we have shown that the DST1 and DST2 gene products, acting in trans in the DST-dependent degradation pathway, are involved in the degradation of AtFer1. Therefore, in addition to the transcriptional regulation described so far, iron is also involved in DST-dependent post-transcriptional regulation of AtFer1 expression. In conclusion, my work has shown that Arabidopsis ferritins are essential elements, which prevent iron toxicity by maintaining a proper labile iron level into the cell, and that sophisticated mechanisms, involving transcriptional and post-transcriptional regulations, permit the tight adjustment of the ferritin accumulation required for the optimal effectiveness of this systemMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Ferritins and iron accumulation in plant tissues

International audienc

Iron utilization and metabolism in plants.

The solubilization and long-distance allocation of iron between organs and tissues, as well as its subcellular compartmentalization and remobilization, involve various chelation and oxidation/reduction steps, transport activities and association with soluble proteins that store and buffer this metal. Maintaining iron homeostasis is an important determinant in building prosthetic groups such as heme and Fe-S clusters, and in assembling them into apoproteins, which are major components of plant metabolism. Such processes require complex protein machineries located in mitochondria and plastids. An essential role for iron metabolism and utilization in plant productivity is evidenced by the strong iron requirement for proper photosynthetic reactions

GSH threshold requirement for NO-mediated expression of the Arabidopsis AtFer1 ferritin gene in response to iron.

International audienceIron treatment of Arabidopsis cultured cells promotes a rapid NO burst within chloroplasts, necessary for up-regulation of the AtFer1 ferritin gene expression. The same occurs in Arabidopsis leaf chloroplasts, and is dependent upon the GSH content of plants. A leaf GSH concentration threshold between 10 and 50 nmol GSHg(-1) FW is required for full induction of AtFer1 gene expression in response to iron