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Ligand competition with a number of chromophoric probes were used to determine the binding affinities for [Zn.sup.II] and [Cu.sup.I] of each metal-binding domain (MBD). The obtained information highlighted the potential toxicity of [Cu.sup.+] in vivo and more sensitive magnitude of copper sensor proteins compared to zinc sensor proteins.

• The Zn/Cd-transporting ATPases, HMA2 and HMA4, essential for root-to-shoot Zn translocation, are also able to transport Cd. Phytochelatins (PCs) are a major mechanism of Cd detoxification through the sequestration of PC-Cd complexes in vacuoles. The roles of HMA2 and HMA4 in root-to-shoot Cd translocation and Cd tolerance were investigated in the PC-deficient, cad1-3 mutant and CAD1 backgrounds. • Six lines, with all possible combinations of hma2, hma4 and cad1 mutations, were constructed. The lines were tested for Cd-sensitivity on agar medium, and radioactive 109Cd was used to measure Cd uptake and translocation from root to shoot over periods of up to 6 d. • In hma4 and hma2,hma4, but not hma2, root-to-shoot Cd translocation was decreased to about 60 and 2%, respectively, of that in the wild-type. Cd sensitivity increased approximately twofold in the hma2,hma4 mutant in both CAD1 and cad1 backgrounds. PC deficiency resulted in an increase in shoot Cd concentrations. • The near-complete abolition of root-to-shoot Cd translocation resulting from the loss of function of HMA2 and HMA4 demonstrates they are the major mechanism for Cd translocation in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

• The Zn/Cd-transporting ATPase, HMA2, has N- and C-terminal domains that can bind Zn ions with high affinity. Mutant derivatives were generated to determine the significance of these domains to HMA2 function in planta. • Mutant derivatives, with and without a C-terminal GFP tag, were expressed from the HMA2 promoter in transgenic hma2,hma4, Zn-deficient, plants to test for functionality. • A deletion mutant lacking the C-terminal 244 amino acids rescued most of the hma2,hma4 Zn-deficiency phenotypes with the exception of embryo or seed development. Root-to-shoot Cd translocation was fully rescued. The GFP-tagged derivative was partially mis-localized in the root pericycle cells in which it was expressed. Deletion derivatives lacking the C-terminal 121 and 21 amino acids rescued all phenotypes and localized normally. N-terminal domain mutants localized normally but failed to complement the hma2,hma4 phenotypes. • These observations suggest that the N-terminal domain of HMA2 is essential for function in planta while the C-terminal domain, although not essential for function, may contain a signal important for the subcellular localization of the protein. [ABSTRACT FROM AUTHOR]

Summary Arabidopsis is remarkable for having eight members of the type 1B heavy metal-transporting P-type ATPase subfamily. Sequence analyses indicate that four, two of which may be targeted to plastids, are related to known Cu(I) transporters and contain N-terminal metal-binding site (MBS) motifs similar to those identified in other organisms. The remaining four are more closely related to known divalent cation transporters of prokaryotes. Three of these form a closely related group and are believed to be Zn(II) transporters. These contain a predicted N-terminal MBS that is a variant of those found in Cu transporters in addition to extended C-terminal regions that contain likely metal-binding sequences. Our current limited knowledge of the physiological roles of these transporters is reviewed and their evolutionary relationships are explored, including an hypothesis that some, particularly the putative divalent cation transporters, are derived from horizontal gene transfer events. [ABSTRACT FROM AUTHOR]

Summary:This paper reports that the glutathione (GSH)-deficient mutant,cad2–1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ-glutamylcysteine.In vitroenzyme assays showed that thecad2–1mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. TheCAD2locus maps to chromosome 4 and is tightly linked to a gene,GSHA, identified by a previously isolated cDNA. A genomic clone ofGSHAcomplements both the phenotypic and biochemical deficiencies of thecad2–1mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that theCAD2gene encodes GCS. Thecad2–1mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of thecad2–1mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.. [ABSTRACT FROM AUTHOR]

Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. The cloning and expression analysis of two genes, AtASD1 and AtASD2, encoding putative alpha-L-arabinofuranosidases in Arabidopsis thaliana are reported here. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Using RT-PCR, RNA gel blot analysis and reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. AtASD1 expression is particularly intense in zones of cell proliferation, the vascular system, developing and regressing floral tissues, and floral abscission zones. By comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones.