Natural variation at the FRD3 MATE transporter locus reveals cross-talk between Fe homeostasis and Zn tolerance in Arabidopsis thaliana.

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

BACKGROUND: Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. RESULTS: A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. CONCLUSIONS: We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

Pathogen Stopping and Metabolism Modulation Are Key Points to Linum usitatissimum L. Early Response against Fusarium oxysporum.

Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.

In situ ESEM using 3-D printed and adapted accessories to observe living plantlets and their interaction with enzyme and fungus.

This paper describes an innovative way of using environmental scanning electron microscopy (ESEM) and the development of a suitable accessory to perform in situ observation of living seedlings in the ESEM. We provide details on fabrication of an accessory that proved to be essential for such experiments but inexpensive and easy to build in the laboratory, and present our in situ observations of the tissue and cell surfaces. Sample-specific configurations and optimized tuning of the ESEM were defined to maintain Arabidopsis and flax seedlings viable throughout repetitive exposure to the imaging conditions in the microscope chamber. This method permitted us to identify cells and tissues of the live plantlets and characterize their surface morphology during their early stage of growth and development. We could extend the application of this technique, to visualize the response of living cells and tissues to exogenous enzymatic treatments with polygalacturonase in Arabidopsis, and their interaction with hyphae of the wilt fungus Verticillium dahliae during artificial infection in flax plantlets. Our results provide an incentive to the use of the ESEM for in situ studies in plant science and a guide for researchers to optimize their electron microscopy observation in the relevant fields.

[A pain assessment logbook]. / Un carnet d'evaluation de la douleur.

Fighting pain is one of nurses' duties. At Dreux general hospital, the medical and nursing staff of the physical medicine and rehabilitation department has gradually put in place since 2004 an information and treatment logbook. It constitutes a way of recording and assessing each patient's pain and consequently facilitates its treatment.

Cytological Approaches Combined With Chemical Analysis Reveals the Layered Nature of Flax Mucilage.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

MuSeeQ, a novel supervised image analysis tool for the simultaneous phenotyping of the soluble mucilage and seed morphometric parameters.

BACKGROUND: The mucilage is a model to study the polysaccharide biosynthesis since it is produced in large amounts and composed of complex polymers. In addition, it is of great economic interest for its technical and nutritional value. A fast method for phenotyping the released mucilage and the seed morphometric parameters will be useful for fundamental, food, pharmaceutical and breeding researches. Current strategies to phenotype soluble mucilage are restricted to visual evaluations or are highly time-consuming. RESULTS: Here, we developed a high-throughput phenotyping method for the simultaneous measurement of the soluble mucilage content released on a gel and the seed morphometric parameters. Within this context, we combined a biochemical assay and an open-source computer-aided image analysis tool, MuSeeQ. The biochemical assay consists in sowing seeds on an agarose medium containing the dye toluidine blue O, which specifically stains the mucilage once it is released on the gel. The second part of MuSeeQ is a macro developed in ImageJ allowing to quickly extract and analyse 11 morphometric data of seeds and their respective released mucilages. As an example, MuSeeQ was applied on a flax recombinant inbred lines population (previously screened for fatty acids content.) and revealed significant correlations between the soluble mucilage shape and the concentration of some fatty acids, e.g. C16:0 and C18:2. Other fatty acids were also found to correlate with the seed shape parameters, e.g. C18:0 and C18:2. MuSeeQ was then showed to be used for the analysis of other myxospermous species, including Arabidopsis thaliana and Camelina sativa. CONCLUSIONS: MuSeeQ is a low-cost and user-friendly method which may be used by breeders and researchers for phenotyping simultaneously seeds of specific cultivars, natural variants or mutants and their respective soluble mucilage area released on a gel. The script of MuSeeQ and video tutorials are freely available at http://MuSeeQ.free.fr.

hca: an Arabidopsis mutant exhibiting unusual cambial activity and altered vascular patterning.

By screening a T-DNA population of Arabidopsis mutants for alterations in inflorescence stem vasculature, we have isolated a mutant with a dramatic increase in vascular tissue development, characterized by a continuous ring of xylem/phloem. This phenotype is the consequence of premature and numerous cambial cell divisions in both the fascicular and interfascicular regions that result in the loss of the alternate vascular bundle/fiber organization typically observed in Arabidopsis stems. The mutant was therefore designated high cambial activity (hca). The hca mutation also resulted in pleiotropic effects including stunting and a delay in developmental events such as flowering and senescence. The physiological characterization of hca seedlings in vitro revealed an altered auxin and cytokinin response and, most strikingly, an enhanced sensitivity to cytokinin. These results were substantiated by comparative microarray analysis between hca and wild-type plants. The genetic analysis of hca indicated that the mutant phenotype was not tagged by the T-DNA and that the hca mutation segregated as a single recessive locus, mapping to the long arm of chromosome 4. We propose that hca is involved in mechanisms controlling the arrangement of vascular bundles throughout the plant by regulating the auxin-cytokinin sensitivity of vascular cambial cells. Thus, the hca mutant is a useful model for examining the genetic and hormonal control of cambial growth and differentiation.