Project Description:

Background: The focus of our project is the Yellow Stripe-Like (YSL) proteins, which are members of the oligopeptide transporter (OPT) family (Yen et al., 2001). YSLs do not appear to transport oligopeptides. Instead, they transport transition metals, like iron and copper, but only when these metal ions are complexed by specific metal chelators produced by plants.

The YSL gene family was identified based on strong sequence similarity to the yellow stripe1 (ys1) gene of maize (Curie et al., 2001). The product of ys1 mediates soil iron uptake in grass species, which acquire iron using a mechanism that is fundamentally different from all non-grass species. In the system used by grasses, iron in the soil is complexed by specific plant-derived Fe(III) chelators called phytosiderophores, which help to solubilize the normally insoluble iron in the rhizosphere. The substrate for YS1 is iron bound by phytosiderophore; un-complexed iron is not transported. Non-grasses neither synthesize nor use phytosiderophores, and so it was surprising to find eight YSL genes in the Arabidopsis genome.

Arabidopsis does make and use a related compound, nicotianamine (NA), which is the biosynthetic precursor to phytosiderophores. Preliminary evidence suggests that YSL proteins mediate transport of metals bound to NA. NA appears to have several roles in plants, all of which relate to allocation of transition metals in plant cells and organs. Several lines of evidence indicate that NA is necessary for distribution of Fe, Zn, and Mn via phloem , and that it is required for transport of Cu in xylem (Pich et al., 1994; Schmidke, I. Stephan, U. W., 1995; von Wiren et al., 1999). NA also appears to maintain intracellular solubility of iron (Becker et al., 1992; Pich et al., 2001), and may help to control oxidative damage (von Wiren et al., 1999). The pleiotropic phenotype of a tomato mutant that lacks NA entirely has even led to the suggestion that nicotianamine could be part of the mechanism plants use to sense iron levels (Becker et al., 1992; Scholz et al. 1992).

Experiments: In this project, a comprehensive description of the function of each YSL protein will be formed. We are determining which metals are transported by each YSL protein using functional complementation in yeast. We are determining the pattern of expression of each YSL gene two ways: promoter fusions to reporters like GUS and/or GFP are providing preliminary information about cell-type and organ specificity. Later, either fusions of full length genes to GFP, or immunofluorescence microscopy will provide more complete expression information. By knowing the details of expression for each YSL, we will understand which cells are active in transporting particular metals. We are also examining whether nutrient conditions affect expression of each YSL family member, to improve our understanding of the environmental conditions that cause mobilization metals within plants. Finally, we are identifying T-DNA insertions in, or creating RNAi constructs directed towards, each YSL gene. The knock-out mutants we are identifying will then be examined for gross defects (symptoms of metal deficiency/oversupply) and tested for more subtle phenotypes under nutrient stress conditions. The total metal content of the mutants will also be determined. By integrating these specific pieces of information for all eight YSL genes, our understanding of the metal ion allocation mechanisms used by plants will be greatly improved.

References:

Becker, R., et al. (1992) Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lysopersicon exculentum Mill.). I. Determination of the apoplasmic and symplasmic iron pools in roots and leaves of the cultivar Bonner Beste and its nicotianamine-less mutant chloronerva. Planta 187, 48-52

Curie, C., et al. (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409, 346-349

Pich, A., et al. (2001) Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration? Planta 213, 967-976

Pich, A., et al. (1994) Iron dependent changes of heavy metals, nicotianamine, and citrate in different plant organs and in the xylem exudate of two tomato genotypes. Nicotianamine as possible copper translocator. Plant and Soil 165, 189-196

Schmidke, I. and Stephan, U. W. (1995) Transport of metal micronutrients in the phloem of castor bean (Ricinus communis) seedlings. Physiologia Plantarum, 106: 147-153

Scholz, G. B., R. Pich, A. Stephan, U.W (1992) Nicotianamine--a common constitutent of strategies I and II of iron acquisition by plants: a review. J. Pl. Nutr. 15, 1647-1665

von Wiren, N., et al. (1999) Nicotianamine chelates both Fe[III] and Fe[II]. Implications for metal transport in plants. Pl. Physiol. 119, 1107-1114

Yen, M.-R., et al. (2001) Maize Yellow Stripe1, and iron-phytosiderophore uptake transporter, is a member of the oligopeptide transporter (OPT) family. Microbiology 147, 2881-2883