Arabidopsis thaliana has eight genes encoding members of the type 1 B heavy metal-transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter-and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.

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Chemically reduced bovine serum albumin (BSA) has been used to modify the surface of water-soluble CdTe quantum dots (QDs). It is demonstrated that the denatured BSA (dBSA) can be conjugated to the surface of CdTe QDs and thereby efficiently improve the chemical stability and the photoluminescence quantum yield (PL QY) of the QDs. It is inferred that a shell-like complex structure CdTe(x)(dBSA)(1-x) will form on the surface of the CdTe "core", resulting in the enhancement of PL intensity and the blue shift of the PL peak. This study of the effects of pH and dBSA concentration on optical properties of dBSA-coated QDs suggests that, at pH 6-9, the solution of dBSA-coated CdTe QDs can keep substantial stability and fluorescent brightness, whereas further increase of pH value leads to a dramatic decrease in PL QY and chemical stability. On the other hand, too high or too low initial dBSA concentration in the QD solution results in a decrease of PL QY for dBSA-coated CdTe QDs. This study provides a new approach of preparing stable water-soluble QDs with high PL QY and controllable luminescent colors for biological labeling applications.

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A unique subfamily of calmodulin-dependent Ca 2؉ -ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca 2؉ -ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca 2؉ -ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg 2؉ , ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca 2؉ -ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals. Ca2ϩ is thought to function as an important second messenger in all eukaryotes (Bootman and Berridge, 1995;Clapham, 1995). In addition, Ca 2ϩ is required for the stability and activity of many proteins and appears to play a critical role in protein processing in the secretory pathway (Rudolph et al., 1989; Gill et al., 1996) Type IIA and IIB pumps include the "ER-type" and the "PM-type" Ca 2ϩ pumps, respectively, first characterized in animal cells. Previously, homologs of ER-or PM-type pumps were distinguished by three criteria: (a) localization to either the ER or PM, respectively, (b) differential sensitivity to inhibitors (e.g. ER-type inhibition by cyclopiazonic acid and thapsigargin), and (c) direct activation of PM-type pumps by calmodulin. However, not all plant homologs conform to these criteria (Bush, 1995;Evans and Williams, 1998).In plants several genes encoding type IIA pumps (ERtype homologs) have been cloned, including LCA1 from tomato (Wimmers et al., 1992), OsCA from rice (Chen et al., 1997), and ACA3/ECA1 (Arabidopsis Ca 2ϩ -ATPase, isoform 3/ER-Ca 2ϩ -ATPase isoform 1) from Arabidopsis (Liang et al., 1997). Consistent with the criteria for a typical ER-type pump, ACA3p (ACA isoform 3 protein) appears to reside in the ER (Liang et al., 1997). However, non-ER locations have been suggested for other isoforms. For example, Ferrol and Bennett (1996) obtained evidence for tonoplast and PM isoforms from membrane fractionation and immunodetection of pumps cross-reacting with an anti-LCA1 antibody.Three plant genes encoding type IIB pumps (PM...

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.Kernel development in maize (Zea mays) proceeds through a series of tightly regulated, overlapping stages. After double fertilization, during the prestorage phase, two distinct cell types are established: the triploid endosperm and the diploid embryo. Despite clearly different cell fates, the embryo and endosperm both rely upon photosynthate from source leaves transported through the maternal pedicel region of the developing kernel, ending at the terminal phloem cells. The presence of Suc-hydrolyzing enzymes, which produce hexose sugars from Suc, have been identified as critical for the establishment of the prestorage phase of seed development, and Suc hydrolysis is an important component of realizable plant yield (Cheng and Chourney, 1999;Weschke et al., 2003). The "invertase control hypothesis," largely based on work from dicots (Wobus and Weber, 1999), is supported in maize by the presence of a cell wall invertase, INCW2, localized to the basal endosperm transfer layer (Talercio et al., 1999). Mutations in this gene result in miniature kernels (the mn1 mutation) and have a severely reduced endosperm (Cheng et al., 1996;Vilhar et al., 2002). Recently, the association of a IVR2, a soluble invertase expressed during early kernel development, with seed yield under conditions of limiting photosynthesis suggests that soluble invertases also play a significant role in providing hexose sugars to support cell division during the prestorage phase (Andersen et al., 2002), as has been previously suggested (Zinselmeier et al., 1999).Invertases exhibit complex regulation at the transcriptional and posttranscriptional levels in response to developmental, environmental, and carbohydrate signals (Sturm, 1999). In addition, small (Ͻ20-kD) inhibitor proteins (INVINH) have been associated with invertase preparations in a number of dico...

The first chelating bis(N-heterocyclic silylene)xanthene ligand [Si(Xant)Si] as well as its Ni complexes [Si(Xant)Si]Ni(η-1,3-cod) and [Si(Xant)Si]Ni(PMe) were synthesized and fully characterized. Exposing [Si(Xant)Si]Ni(η-1,3-cod) to 1 bar H at room temperature quantitatively generated an unexpected dinuclear hydrido Ni complex with a four-membered planar NiSi core. Exchange of the 1,3-COD ligand by PMe led to [Si(Xant)Si]Ni(PMe), which could activate H reversibly to afford the first Si-stabilized mononuclear dihydrido Ni complex characterized by multinuclear NMR and single-crystal X-ray diffraction analysis. [Si(Xant)Si]Ni(η-1,3-cod) is a strikingly efficient precatalyst for homogeneous hydrogenation of olefins with a wide substrate scope under 1 bar H pressure at room temperature. DFT calculations reveal a novel mode of H activation, in which the Si atoms of the [Si(Xant)Si] ligand are involved in the key step of H cleavage and hydrogen transfer to the olefin.