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Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus  laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd2+) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd2+-sensitive phenotype. Further analyses showed that Cd2+ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd2+ tolerance.

Understanding the functional connections between genes, proteins, metabolites and mineral ions is one of biology's greatest challenges in the postgenomic era. We describe here the use of mineral nutrient and trace element profiling as a tool to determine the biological significance of connections between a plant's genome and its elemental profile. Using inductively coupled plasma spectroscopy, we quantified 18 elements, including essential macro- and micronutrients and various nonessential elements, in shoots of 6,000 mutagenized M2 Arabidopsis thaliana plants. We isolated 51 mutants with altered elemental profiles. One mutant contains a deletion in FRD3, a gene known to control iron-deficiency responses in A. thaliana. Based on the frequency of elemental profile mutations, we estimate 2-4% of the A. thaliana genome is involved in regulating the plant's nutrient and trace element content. These results demonstrate the utility of elemental profiling as a useful functional genomics tool.

SUMMARYThe plant vacuole is an important organelle for storing excess iron (Fe), though its contribution to increasing the Fe content in staple foods remains largely unexplored. In this study we report the isolation and functional characterization of two rice genes OsVIT1 and OsVIT2, orthologs of the Arabidopsis VIT1.

Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. However, phytochelatins (PCs) are generally considered to function as intracellular heavymetal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase (Adh) promoter (Adh:: TaPCS1͞cad1 C admium is a widespread nonessential toxic heavy metal, released into the biosphere mostly by modern industry (1). Phytochelatins (PCs) play an essential role in heavy-metal detoxification in plants and fungi (2, 3). PCs chelate heavy metals and then PC-metal complexes are translocated across the tonoplast and sequestered in vacuoles (4, 5), thus decreasing the heavy-metal content in the cytosol of yeast and plant cells. PCs are synthesized from glutathione by the enzyme PC synthase (PCS) (6-9). However, PC research has focused mainly on PC transport into vacuoles (4, 5), and it remains unknown whether long-distance transport of PCs or PCS occurs in plants.Cd 2ϩ is taken up by plants, and plants have been proposed to provide an efficient system for heavy-metal removal from soils (10, 11). Soil composition affects Cd 2ϩ sensitivity. For example, silicon in soil reduces heavy-metal sensitivity (12, 13). Normally, Cd 2ϩ concentrations in roots are at least 10 times greater than those in shoots (14). However, for efficient phytoextraction from soils, heavy metals must be translocated into aerial tissues for later harvest. Translocation of Cd 2ϩ from roots to shoots has been studied in diverse plant species (15-18) and has been proposed to occur via the xylem of Indian mustard in a PCindependent manner (16). The molecular mechanisms for rootto-shoot Cd 2ϩ transport remain largely unknown, and PCs are predicted not to undergo long-distance transport but to enhance vacuolar heavy-metal sequestration in the cells in which they are produced.The cad1-3 mutant is a recessive, loss-of-function mutation in the Arabidopsis AtPCS1 gene (7) (9) by PCR. The TaPCS1 PCR fragment was subcloned into pBluescript II SK(ϩ) vector. A 3ϫ myc tag DNA sequence was constructed by PCR recovery from a plasmid containing c-myc and then subcloned into the pGEM-T Easy vector (Promega). The subcloned myc fragment was recovered with SpeI and SacI enzymes and then fused to the 3Ј end of the TaPCS1 ORF in the pBluescript II SK(ϩ) vector at the SpeI site. All PCR products were confirmed by sequencing (Retrogen, La Jolla, CA). The fusion sequence was then digested with BamHI and SacI and subcloned into the binary expression vector pBI121, either with the cauliflower mosaic virus 35S or the Arabidopsis alcohol dehydrogenase (Adh) promoter (20). Both constructs were transformed into the PC-deficient Arabidopsis mutant cad1-3 by direct Agrobacterium tumefaciens-mediated transformation using the floral dip technique (21). T2 seeds with 3:1 segregation on kanamycin plates were used for hom...

Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.

Nitrate reallocation to plant roots occurs frequently under adverse conditions and was recently characterized to be actively regulated by Nitrate Transporter1.8 (NRT1.8) in Arabidopsis (Arabidopsis thaliana) and implicated as a common response to stresses. However, the underlying mechanisms remain largely to be determined. In this study, characterization of NRT1.5, a xylem nitrate-loading transporter, showed that the mRNA level of NRT1.5 is down-regulated by salt, drought, and cadmium treatments. Functional disruption of NRT1.5 enhanced tolerance to salt, drought, and cadmium stresses. Further analyses showed that nitrate, as well as Na + and Cd 2+ levels, were significantly increased in nrt1.5 roots. Important genes including Na + /H + exchanger1, Salt overly sensitive1, Pyrroline-5-carboxylate synthase1, Responsive to desiccation29A, Phytochelatin synthase1, and NRT1.8 in stress response pathways are steadily up-regulated in nrt1.5 mutant plants. Interestingly, altered accumulation of metabolites, including proline and malondialdehyde, was also observed in nrt1.5 plants. These data suggest that NRT1.5 is involved in nitrate allocation to roots and the consequent tolerance to several stresses, in a mechanism probably shared with NRT1.8.

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers. 1 p53 is a sequence-specific transcription factor and plays a critical role in activating the expression of genes involved in cell cycle arrest or apoptosis under conditions of genotoxic stress. 2,3 For over two decades, p53 was thought to be the only gene of its kind in the vertebrate genomes. This strong conviction, which was widely accepted in the p53 field, has now been proven to be incorrect. Two genes, referred to as p63 and p73, have been found to encode proteins that share a significant amino-acid identity in the transactivation domain, the DNA binding domain, and the oligomerization domain with p53. In the short period since their cloning, a number of investigators have reported on the structure, the function and the regulation of p63 and p73. This review summarizes the current information on the p63 and the p73 genes, with a focus on the differences between the three members in this newly defined p53-gene family.Keywords: p73; p53; c-Abl; apoptosis Abbreviations: HPV-16, human papilloma virus-16; IGFBP, Insulin-like growth factor binding proteins; IR, ionizing irradiation; MEFs, mouse embryo ®broblasts; MMS, methylmethane sulfonate; OD, oligomerization domain; SAM, sterile alpha motif; TA, transactivation domain; DN p63, N-terminal deleted p63 variants Alternative splicing of p63 and p73The p53 gene generates a single mRNA with a single open reading frame. In contrast, both the p63 and the p73 genes generate several differentially spliced variants. With the p73 gene, alternative splicings not only add or delete coding sequences, but also alter the reading frame. Hence, the p63 and the p73 genes can each encode several different proteins. Most notably, both the p63 and the p73 genes encode alternatively spliced C-terminal regions that are not found in the p53 protein (Figure 1).The p63 gene encodes at least six open reading frames: from the usage of two different promoters/ATG in combination with three alternatively spliced C-terminal ends (Figure 1). The three isoforms (TA-a, TA-b and TAg) are produced by a 5'-promoter and alternative splicing at the 3' end of the gene. These three isoforms contain the coding sequence for the N-terminal transactivation (TA) domain. Each of these three splice variants can also be expressed from an internal promoter upstream of exon 3', that provides a different ATG to initiate translation downstream of the TA domain. These N-terminal deleted p63 isoforms are referred to as DN-alpha, DN-beta and DNgamma. These DN p63 isoforms do not activate transcription but instead can inhibit the transactivation functions of the full length p63 proteins and of p53. 4 The p73 gene generates at least six open reading frames with alternatively spliced 3-region. Initially, two isoforms of p73 were identified: 5 the full length alphaisoform and a C-terminal shortened beta-isoform resulting from the alternative splicing of exon 13. Four other spliced forms of p73 have since been identified in...