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A complete inventory of metallochaperone-like proteins containing a predicted HMA domain in Arabidopsis revealed a large family of 67 proteins. 45 proteins, the HIPPs, have a predicted isoprenylation site while 22 proteins, the HPPs, do not. Sequence comparisons divided the proteins into seven major clusters (I-VII). Cluster IV is notable for the presence of a conserved Asp residue before the CysXXCys, metal binding motif, analogous to the Zn binding motif in E. coli ZntA. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 in Cluster IV were studied in more detail. All but HIPP21 could rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and atx1 mutants. In Arabidopsis, single and double mutants did not show a phenotype but the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type suggesting the HIPPs can have a role in Cd-detoxification, possibly by binding Cd. Promoter-GUS reporter expression studies indicated variable expression of these HIPPs. For example, in roots, HIPP22 and HIPP26 are only expressed in lateral root tips while HIPP20 and HIPP25 show strong expression in the root vasculature.

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.

* The usefulness of the zinc (Zn)-fluorophore, Zinpyr-1, to examine the localization of Zn in the roots of Arabidopsis has been investigated. * In wild-type roots Zinpyr-1 fluorescence was predominantly in the xylem. The fluorescence signal was abolished by the application of the Zn-chelator, N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and was increased by increasing exogenous Zn in the medium, indicating that fluorescence reflected relative Zn concentrations. * In the hma2, hma4 double mutant, which is deficient in root to shoot Zn translocation, Zinpyr-1 fluorescence was low in the xylem and high in the adjacent pericycle cells in which HMA2 and HMA4 are specifically expressed in a wild type. Zinpyr-1 fluorescence was also increased in the endodermis. * These results show that Zinpyr-1 can be used to examine the effects of mutations in Zn transporters on the localization of Zn in Arabidopsis roots and should be a useful addition to the tools available for studying Zn homeostasis in plants.

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.

The hydrolysis of sucrose by cell-wall invertases (cwINV) and the subsequent import of hexoses into target cells appears to be crucial for appropriate metabolism, growth and differentiation in plants. Hexose uptake from the apoplast is catalysed by monosaccharide/H+ symporters (Sugar Transport Proteins or STPs), which have the potential to sense sugars. Import of extracellular hexoses may generate signals to orchestrate cellular activities, or simply feed metabolic pathways distinct from those fed by sucrose. It is predicted that Arabidopsis has six cwINV genes and at least 14 STP genes. These genes show different spatial and temporal patterns of expression, and several knock-out mutants have been isolated for analysis. AtSTP1 transports glucose, galactose, xylose, and mannose, but not fructose. It accounts for the majority of the AtSTP activity in vegetative tissues and its activity is markedly repressed by treatment with exogenous sugars. These observations are consistent with a role in the retrieval of cell-wall-derived sugars, for example, during carbohydrate limitation or cell expansion. The AtSTP1 gene is also expressed in developing seeds, where it might be responsible for the uptake of glucose derived from imported sucrose. The large number of AtcwINV and AtSTP genes, together with complex patterns of expression for each, and the possibility that each protein may have more than one physiological function, provides the plant with the potential for a multiplicity of patterns of monosaccharide utilization to direct growth and differentiation or to respond flexibly to changing environmental conditions.

Summary3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid β-oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T-DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long-chain (C16 to C20) fatty acyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long-chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal β-oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies. [ABSTRACT FROM AUTHOR]