Manganese uptake in Arabidopsis : genomic analysis and regultory networks – MANOMICS

By combining approaches such as genetics, biochemistry (Partner 1) and genomics (Partner 2), we are attempting to decipher the crucial steps of Mn entry in the root and its regulation. 1) In particular, we are studying the respective role of the metal transporters NRAMP1 and IRT1 in the transport of Fe and Mn at the root surface and by extension, the interaction between Fe and Mn homeostases. To that aim, gene expression in plants is tentatively modified (production of mutants and transgenic overexpressor lines) and the resulting effect on metal transport analysed (Partner 1). 2) In order to identify new actors of Mn homeostasis, a global analysis of the response of the plant to Mn deficiency is carried out by techniques such as transcriptomics or proteomics (Partner 2). AMong the genes/proteins whose abundance is modified upon the treatment, we will focus on those that are likely to play a role in the regulation of Mn uptake (transcription factors, Kinases/Phosphatases,…). Their function in the maintain of Mn homeostasis will then be evaluated by reverse genetics, that is by inactivating their gene and testing the impact on Mn uptake in roots (Partners 1 and 2).

The production of tools for the analysis of NRAMP1 and IRT1 is under way (plasmids, double mutants, overexpressor lines, etc…) (Partner 1).
Transcriptmic data to describe Arabidopsis transcripts response to Mn deficiency have been obtained in wild-type as well as in the Mn transport defective mutant nramp1. Data analysis in under way (Partner 2).

The physiological relevance of Mn uptake in plants has been so far controversial and greatly underestimated. The accepted dogma among animal and plant biologists has been that the physiological requirement for Mn is lower than their uptake capacity. Recently however, Mn availability was shown to greatly limit plant growth unless the specific high-affinity Mn transporter NRAMP1 is activated (Partner 1). More importantly, overproduction of NRAMP1 in the plant significantly increases its biomass. Given the enhanced demand for increased plant biomass production, extending our knowledge on the processes involved in Mn uptake in plants is a first step in the exploration of breeding approaches, in collaboration with plant breeders, to develop Mn-efficient germplasms.

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Manganese (Mn) is an essential nutrient in virtually all organisms. In plants, Mn deficiency is a widespread nutritional disorder that causes substantial yield losses and decreases the quality of edible plant parts. Despite its importance, little is known with regard to the uptake of Mn by plant roots and the molecular mechanisms that control cellular Mn homeostasis. In this proposal, we attempt to dissect the role of two plasma membrane bound transport proteins, the Natural Resistance-Associated Protein 1 (NRAMP1) and the Iron-Regulated Transporter 1 (IRT1) in the uptake of Mn and iron (Fe), which are thought to compete for binding sites at these transporters. The French cooperating group (P1) has generated transgenic lines in which either one or a combination of both transporters is knocked out or ectopically expressed, which will allow for dissecting the respective roles of these transporters in Fe and Mn uptake. By using a top-down high-throughput approach, the Taiwanese cooperating group (P2) further attempt to conduct a state-of-the-art analysis of changes in the transcriptome and proteome profile upon Mn deficiency in the model plant Arabidopsis, which will yield the first thorough description of the Arabidopsis Mn deficiency response and which will further serve as a basis for a comprehensive reverse genetic screening aimed at identifying key players in cellular Mn homeostasis. Transcriptional profiles will be analyzed by RNA sequencing (RNAseq) on the Solexa II platform, protein profiles will be analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with high-energy collision dissociation. Reverse genetic experiments, conducted by the Taiwanese lab, will be guided by constructing coexpression and protein interolog networks, assuming that central hubs in these networks may play critical roles. As a further objective we seek to understand the molecular mechanisms that control Mn homeostasis in Arabidopsis. In a forward genetic screen, the Taiwanese lab has identified a mutant that is defective in inducing the phenotype typical of Mn-deficient plants (manic, manganese deficient root hair defective), based on the inhibition of root hair elongation by high light conditions which is overcome by Mn deficiency. manic plants show deregulated Mn uptake in the roots while the uptake of other metals does not deviate from the wild-type. The mutated gene has been cloned and shown to encode a protein related to the human mediator of RNA polymerase transcriptional regulation (MED6). Assuming a potential key role of MED6 in recalibrating cellular Mn homeostasis, we plan to identify interacting partners by yeast-two hybrid screening. Finally, regulation of IRT1 and NRAMP1 gene expression by Mn will be studied by the French partner. The bHLH transcription factor FIT controls IRT1 and NRAMP1 expression in response to Fe deficiency. We will investigate the role of FIT in their Mn response as well as the possibility that they are additionally post-translationally regulated by Mn availability. Candidate interacting partners of NRAMP1, identified by split-ubiquitin screening, will be tested by means of reverse genetics, biochemistry and cell biology.
The combination of these results will help understand the complex interplay of Mn and Fe, will generate a database for genes and proteins that are differentially expressed upon Mn deficiency, and will allow insights into the mechanisms controlling Mn uptake. A long-term goal of this project is to provide the basis for collaborations between plant breeders and physiologists to develop plants that are better adapted to Mn deficiency.