Transport, abiotic stress and root development - Responsible: Nathalie Verbruggen (P3) /Marc Boutry (P4A)
The root plays a major role in mineral nutrient and water acquisition. It is also the first organ to undergo abiotic stress caused by either pollutants or deficit of nutrients. These various stresses as well as pH, osmotic and drought stress might have tremendous effects on the root architecture although the molecular links are still poorly deciphered. More particularly, transporters involved in water and nutrient transport have been identified at the molecular level. However, how they intervene in the root development, how they are regulated and how they respond to abiotic stress is still largely unknown. This work package is dedicated to a better molecular identification of the relationship between root development, nutrition and abiotic stress. Among the different mechanisms underlying adaptation to metal toxicity/nutrient deficiency, a focus will be made on transport processes (at the cellular level and between root and shoot) and their regulation. Some partners have already characterized at the gene and protein level several key transporters active in root tissues. They belong to three transporter families: P-type ATPases (P3, P4A), aquaporins (P4B) and ATP Binding Cassette (ABC) transporters (P4A). We will therefore build on this knowledge to address the regulation and function of these transporters in relationship with root development. While the project proceeds, other transporters identified in this or other work packages will be integrated in this analysis.
This WP will benefit from proteomics approaches (P4) for the identification of root proteins that are differentially expressed or post-translationally modified upon stress. In addition, P1 will provide FACS facilities to study cell-type specific responses to stress. Of particular significance here are the specific responses in the epidermal tissue in direct contact with the soil and the vascular tissues, which mediate the communications between the root and the shoot. In addition, as a tool to investigate the kinetics of root-shoot communication in stress, P1D will offer a robotized imaging platform for real time follow-up of the effect of stress applied to the roots, on the key physiological parameters in the shoot tissue, i.e. photosynthesis and transpiration. P1D has established a unique lab-scale robotized setup with combined thermography, chlorophyll fluorescence imaging and video imaging. Procedures for time-lapse photography and automated generation of movies for rapid visualisation of changes, as well as for automatic quantification of leaf surface temperature and chlorophyll fluorescence intensity are available.
How particular stress conditions will interfere with cell division will be studied in collaboration with P1 and cell expansion with P5. Firstly kinematic analyses performed in collaboration with P1C will enable to pinpoint the contribution of cell cycle and cell expansion parameters in the growth response to the stress. In collaboration with P1A the effect on lateral root initiation and root architecture will be charted. For further characterization of cell cycle effects P1B can contribute by analysing cell cycle gene activities by means of a collection of cell cycle promoter GUS lines that have been generated in his group. Differences in cell expansion parameters can be further characterized using extron measurements and FESEM technology in the lab of P5.
Particular transporters or stress conditions will be studied as follows. |
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H+-ATPases Plasma membrane H+-ATPase creates the proton-motive force that activates many secondary transporters and thus controls many aspects of mineral nutrition. Transgenic tobacco plants that do not express a H+-ATPase subfamily any more showed profound modifications of their development. We (P4A) wish to recapitulate this approach in Arabidopsis, using cell-type specific promoters (collaboration with P1), to drive H+-ATPase expression extinction and so determine locally the specific role of this pump. In parallel, we will disturb the cell and organ development by expressing a H+-ATPase isoform that has been constitutively activated by deleting the C-terminal auto-inhibitory domain. This approach was recently implemented using a constitutive transcription promoter and led to developmental disturbances such as leaf epinasty and stem twisting. Here also, using specific transcription promoters (interaction with P1A,B,C) should allow us to determine the role of this pump in different root cell tissues. The physiological consequences (e.g. root proton pumping) of the constitutive H+-ATPase activation will be determined in collaboration with P5 (see WP2.2.)
Finally, this pump is regulated by phosphorylation of the penultimate Thr and binding of regulatory 14-3-3 proteins, resulting in an activated complex. Two other phosphorylation sites that negatively regulate the enzyme have been recently discovered (P5A, unpublished data). While the basic mechanisms of these regulatory features will be deciphered, P4A will determine how they are involved in the root development or associated with stress response, using specific antibodies that discriminate between phosphorylated isoforms. How these regulatory features are integrated in the root development or associated with stress-response is currently unknown and will be studied by expressing site-directed mutant proteins in transgenic plants and characterizing the growth response by means of kinematic analysis (P1C).
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Aquaporins
Aquaporins are involved in the regulation of transcellular water flow for long-distance transport and in osmotic adjustment within a cell and between the cytoplasm and the cell wall space during, for instance, cell elongation. These transporters are highly expressed in primary root tips and particularly in zones of cell elongation and their activity is regulated by phosphorylation, heteromerization and cellular factors such as pH and Ca++.
To determine aquaporin function in growth processes, P4B will obtain plant lines with deregulated aquaporin gene expression and analyze the root growth and water-related parameters. P4B has developed a biophysical method to measure accurately the osmotic water permeability of plant protoplasts. The effect of water stress on cell water permeability can be determined. We also plan to set up a pressure chamber and a cell pressure probe system to analyze the root water conductivity and the water permeability of cells within the roots. The regulation of aquaporin by phosphorylation and heteromerization during root growth and development will be also investigated. In the group of P1D a number of PIP2 aquaporin family members were found to be negatively regulated by ethylene, and associated with short-celled tissues. Hence these are good candidates for future research on root growth in a hormonal context (P1D, link to WP2 and WP3).
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Last Updated on Friday, 08 February 2008 15:43 |
Nutrient deficiency Mineral nutrients are one of the prime limiting factors for plant growth and development.
Deficiencies of essentials macronutrients are know to elicit various molecular responses, resulting in modification of the root system architecture and the shoot to root biomass ratio. We will analyze in more detail two cases that are studied by P3 and P4A.
5.4.1. Mg deficiencyIt is not clearly identified what systems mediate Mg transport into the root or how Mg is loaded into and unloaded from the vascular system. The overall goal is to understand how plants acquire Mg through the roots, distribute and regulate their internal Mg level. Two complementary approaches will be used to optimise our chances to elucidate these mechanisms: (1). to clone new genes in Arabidopsis mutants that contain abnormal levels of Mg. (2). to identify transcriptome changes in roots in response to Mg deficiency.
The focus of the first objective will be the study of Mg hypo-accumulators and hyper-accumulators listed in the Arabidopsis Ionomics Database (NSF Plant Functional Genomics). These mutants are a novel tool for the studies in the IAP project. Our analysis will include a physiological characterisation consisting of photosynthesis measurements using fluorescence imaging and infrared gas analysis (collaboration with P1D), mineral and sugar profiles of root and shoot, phloem and xylem sap composition analyses (interaction with P2). P3 will use both positional cloning and microarray-based cloning to identify the mutations in genes responsible for Mg-profile changes. Anticipated results of this forward genetic approach are to discover novel genes involved in Mg transport and/or homeostasis.
The goal of the second proposed study is to provide a better understanding of Mg uptake in roots, allocation and homeostasis in plants. P3 recently reported the first physiological analysis of Mg deficient Arabidopsis. This study provides a concrete basis for further genetic analysis, using a genomic approach. The roots, which first undergo external Mg limitation, will constitute the material for transcriptomic analysis using Arabidopsis microarrays (in collaboration with P1).
5.4.2. Fe deficiencyFe deficiency has already been characterized in more detail by many laboratories. However, P4A recently discovered that PDR3, an ATP-binding cassette (ABC) transporter belonging to the Pleiotropic Drug Resistant subfamily, was strongly induced under iron deficiency conditions. This expression was recently localized to the root (unpublished data). As iron transporters have already been identified, we favour the hypothesis that the ABC transporter might be involved in transporting out of the epidermal cell molecules such as organic acids that might improve iron solubility in the soil. Alternatively, PDR3 might play a role in parenchyma cells associated with conducting vessels by transporting outside the cell metabolites that keep iron soluble. This hypothesis will be tested using different tools. PDR3 expression will be determined at the cell level using the GUS reporter. Functional aspects will be analyzed by examining transgenic plants in which PDR3 expression has been prevented by gene knock-out or RNA interference. This analysis will be performed in collaboration with P1.
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