Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of~55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd 2ϩ than controls. PCS expression mediates Cd 2ϩ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd 2ϩ and Cu 2ϩ and are unable to synthesize phytochelatins upon Cd 2ϩ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.

In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde EDTA, 0.1% Nonidet P-40, 25% glycerol, 5% sucrose, 25 ,g of cycloheximide per ml). After successive steps of centrifugation at 5,000 and 12,000 rpm, 20 ml of the final supernatant (S-12) was used for HSG preparation (see above) and 10 ml was used for preparation of the polysomal and postpolysomal fractions. To this end, Nonidet P-40 was added to 0.5% (final concentration) and each 5 ml of S-12 was layered on top of a 2-ml cushion containing 20 mM Tris hydrochloride (pH 7.8), 500 mM KCI, 5 mM MgCl2, 1 mM EDTA, 0.5% Nonidet P-40, 25% glycerol, 10% sucrose, and 25 ,ug of cycloheximide per ml. Polysomes plus HSGs were sedimented by centrifugation at 171,000 x g (70.1 Ti rotor for 1 h). The polysomal messenger ribonucleoprotein (mRNP) was solubilized from the pellet by extraction with buffer P

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of -17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40°C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25°C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.All types of eucaryotic organisms respond to supraoptimal temperatures by synthesis of heat shock proteins (hsps) (1,5,23,24), which are evidently essential for heat shock survival and recovery (2,14,16,20,23

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