Zinc is a constituent of several enzymes: carbonic anhydrase dehydrogenases; aldolases; Cu/Zn superoxide dismutase; isomerases; transphosphory-lases; and RNA and DNA polymerases. Therefore, Zn deficiency results in malfunction or no function of these enzymes. Zn-metalloproteins (Zn-finger motif) are regulators of gene expression (DNA-binding transcription factors). In the absence of these, RNA polymerase cannot complete its function of transcribing genetic information from DNA into RNA. Zinc is a cofactor of more than 200 enzymes, such as oxidoreductases, hydrolases, transferases, lyases, isomerases, and ligases. Many of the metalloenzymes are involved in the synthesis of DNA and RNA and protein synthesis and metabolism.
Metal-transporting genes have been identified in Arabidopsis (Brassicaceae). Overexpression of an Arabidopsis zinc transporter cation diffusion facilitator (CDF) gene enhanced resistance to Zn accumulation. Transgenic plants showed increased Zn uptake and tolerance and antisense of this gene led to wild-type Zn tolerance in transgenic plants. Zinc transporters can be manipulated to increase selectivity and accumulation of metal ions. In Brassicaceae, about 21 species belonging to three genera (Cochlearia, Arabidopsis, and Thlaspi) are reported to be zinc hyperaccumulators. Zhao et al.  isolated and identified the gene ZNT1 as one of the micronutrient transport genes with high sequence homology with other Zn transport genes isolated from yeast.
A family of zinc transporter genes that responds to zinc deficiency has also been identified in Arabidopsis. Zn hyperaccumulation in Thlaspi caerulescens is because of the ZNT1 gene, which encodes a high-affinity Zn transporter. This gene is constitutively expressed at a much higher level in T. caerulescens than in T. arvense, where its expression is stimulated by Zn deficiency. In fact, plant Zn status is shown to alter the normal regulation of Zn transporter genes in T. caerulescens. An important aspect of Zn hyperaccumulation and tolerance in T. caerulescens is also the production of low molecular weight compounds involved in Zn detoxification in the cell (cytoplasm and vacuole) and in the long-distance transport of Zn in the xylem vessels.
Citrate was not shown to play an important role in Zn chelation and malate had constitutively high concentrations in the shoots of the accumulator T. caerulescens and the nonaccumulator T. ochroleucum. More recently, direct measurements of the in vivo speciation of Zn in T. caerulescens using the noninvasive technique of x-ray absorption have revealed that histidine is responsible for the transport of Zn within the cell, whereas organic acids (citrate and oxalate) chelate Zn during long-distance transport and storage. Another constitutive aspect of T. caerulescens is the high Zn requirement for maximum growth, compared to other species. This probably depends on the strong expression of the metal sequestration mechanism, which would subtract a large amount of intracellular Zn to normal physiological processes even when the Zn supply is low.
Several transporters implicated in the uptake of divalent nutrient cations like Ca2+, Fe2+, or Zn2+ appear to be able to transport other divalent cations. For example, heterologous expression in yeast of IRT1 from A. thaliana, an iron-repressed transporter in the ZIP family of metal transporters, suggests a broad-range specificity of transport for Cd2+, Fe2+, Mn2+, Zn2+, and possibly other divalent cations (TC 2.A.5.1).
The expression of IRT1 is strongly induced in plants under conditions of iron deficiency and is repressed in iron-replete plants. This correlates well with the finding that cadmium uptake is enhanced in iron-deficient pea seedlings. However, these transporters are tightly regulated at the transcriptional and post-transcriptional levels, and, to date, no reports on plants engineered to overexpress transporters of the ZIP family have been issued. Tobacco plants engineered to contain increased amounts of NtCBP4 protein (TC 1.A.1.5.1), a putative cyclic-nucleotide and calmodulin-regulated cation channel in the plasma membrane, displayed an increased sensitivity to lead, a 1.5-to 2.0-fold shoot accumulation of lead, and an increased nickel tolerance. Yeast cells expressing the wheat LCT1 cDNA (TC 9.A.20.1.1), encoding a low-affinity cation transporter, were hypersensitive to Cd2+ and accumulated increased amounts of cadmium . Plants overexpressing AtNramp3 (TC 2.A.55), a member of the Nramp family of metal transporters, were hypersensitive to Cd2+, but enhanced cadmium accumulation was not observed.
In phytoremediation, it must be considered that a transporter capable of transporting a specific contaminant metal cation is capable of transporting other competing cations, like Ca2+ or Zn2+, under natural soil conditions if the latter ions are present in large excess. Therefore, it is desirable to better understand what governs the specificity of membrane transporters, in order to generate mutated transporters with altered specificities . Understanding the regulation of ZIP family members in T. caerulescens and analyzing Arabidopsis mutants with altered metal responses will also help to identify novel target genes and strategies for the generation of plants with enhanced metal uptake .
To date, numerous examples have been demonstrated to have the potential for phytoremediation — for example, Pb, Ni, Zn, Al, Se, Au, and As. Arazi et al.  have described a tobacco plasma membrane calmodulin-binding transporter that confers Ni2+ tolerance and Pb2+ hypersensitivity. Zinc-transporter To investigate the in vivo role of this gene, transgenic plants with the ZAT coding sequence exhibited increased Zn resistance and accumulation in the roots at high Zn concentrations .
In maize, zinc accumulation was found to be genetically controlled and affected by additive genes . Four genes were found to be the minimum segregation factors in the (high x low) crosses for Zn accumulation. Zinc deficiency also increases root exudation of amino acids, sugars, and phenolic substances at different degrees in different species .
Three wheat genotypes (Triticum aestivum and T. turgidum) differed in their root-growth response to low zinc levels . The zinc-efficient genotype increased root and shoot dry matter and developed longer and thinner roots (a greater proportion of fine roots with diameter of 0.2 mm) compared with the less efficient genotype. Due to a larger root surface area, the efficiency of zinc uptake increased. In wheat, Zn can be remobilized from leaves under Zn deficiency. Also, spinach, potato, navy bean, tomato, sorghum, and maize show great variations in Zn efficiency [118,119].
Arabidopsis thaliana has multiple Zn transporters designated ZIP1, ZIP2, ZIP3, and ZIP4. Grotz et al.  demonstrated the specificities of each of the ZIP genes with their experiments. They tested other metal ions for their ability to inhibit Zn uptake mediated by these proteins. Zn uptake by ZIP1 was not inhibited by a tenfold excess of Mn, Ni, Fe, and Co. Zn was the most potent competitor, demonstrating that ZIP1 prefers Zn as its substrate over theses metal ions. Cd and Cu also inhibited Zn uptake, but to a lesser extent. This suggests that Cd and Cu may also be substrates for ZIP1.
The ZIP family members have 309 to 476 aa; this range is largely due to variation in the number of residues between transmembrane domains III and IV, a domain designated as "variable." The amino acid sequences of all the known ZIP family members were aligned, and a dendrogram describing their sequence similarities was generated . ZNT1 is 379 aa in length and shares the same structural features exhibited by the other members of the ZIP family, including eight putative transmembrane domains and a highly hydrophilic cytoplasmic region predicted to reside between transmembrane domains three and four. The putative cytoplasmic domain contains a series of histidine repeats, which may define a metal-binding region for the transporter. Zinc transporters can be manipulated to increase selectivity and accumulation of metal ions.
Pence et al.  reported on the cloning and characterization of a high-affinity Zn2+ transporter cDNA, ZNT1, from the Zn/Cd-hyperaccumulating plant, Thlaspi caerulescens. Through comparisons to a closely related, nonaccumulator species, Thlaspi arvense, the researchers determined that the elevated ability of T. caerulescens to take up Zn and Cd was due, in part, to an enhanced level of expression of Zn transporters. Previous physiological studies by the group indicated that the hyperaccumulating ability of T. caerulescens was linked to Zn transport at a number of sites in the plant. The researchers transformed a Zn transport-deficient strain of yeast, ZHY3, with a cDNA library from T. caerulescens. By screening for growth on low-Zn medium, they were able to isolate seven clones, five of which represented a 1.2-kb cDNA designated ZNT1 (for Zn transporter) that restored the yeast's ability to grow under low-Zn conditions. The ZNT1 gene displayed considerable identity to two Arabidopsis thaliana metal transporter genes, ZIP4 (for transporting Zn) and IRT1 (for transporting Fe).
For purposes of comparison, they then cloned the homolog of ZNT1 (designated ZNT1-arvense) from the nonhyperaccumulator species T. arvense. Expression studies using northern blots of RNA isolated from the roots and shoots of both Thlaspi species revealed that ZNT1 is expressed in T. caerulescens at extremely high levels. In contrast, expression of ZNT1 in T. arvense could only be detected at a very low level in shoots and roots, and then only when the plants had been exposed to conditions of Zn deficiency. To further explore the role of Zn status on ZNT1 expression, Pence et al.  exposed both species to a range of Zn concentrations. They found that when T. caerulescens was grown in a nutrient solution containing an excess of Zn (50 |M), the transcript level of ZNT1 decreased, indicating that ZNT1 is not expressed constitutively in T. caerulescens. ZNT1 transcript levels in T. arvense appeared to be unaffected by exposure to excess Zn.
Transport studies in T. caerulescens show that ZNT1 mediates high-affinity Zn transport. In many plant species, the induction of a high-affinity transporter is characteristic of a nutrient-deficiency response and would correlate with the expression pattern observed for ZNT1 in T. arvense.
The authors speculate that the hyperaccumulation phenotype in T. caerulescens may then be due to a mutation in the plant's ability to sense or respond to Zn levels — that is, these plants may be functioning as if they are constantly experiencing Zn deficiency. They propose that this is likely the result of a change in global regulation linked to the plant's overall Zn status; this supports the concept that the Zn hyperaccumulation phenotype, at least in this species, is due to a change in the regulation and not the constitutive expression of a single gene.
Several mutants with altered response to heavy metals have been isolated from A. thaliana. Cadmium-hypersensitive mutants with defects in phytochelatin synthetase and possibly in g-glutamylcysteine synthetase and glutathione synthetase have been isolated by Howden et al. [65,66]. Chen and Goldsbrough  found an increased activity of g-glutamylcysteine synthetase in tomato cells selected for cadmium tolerance. Some of these genes may prove useful in modifying suitable target plants for phytoremediation, although there are doubts about the usefulness of genes involved in phytochelatin synthesis .
Ferritin is an iron storage protein found in animals, plants, and bacteria. It comprises 24 subunits, which may surround in a micellar up to 4500 ferric atoms . It provides iron for the synthesis of iron proteins such as ferredoxin and cytochromes. It also prevents damage from free radicals produced by iron/dioxygen interactions. Ferritin has been found to provide an iron source for treatment of anemia. It was thus proposed that increase of the ferritin content of cereals by genetic modification may help to solve the problem of dietary iron deficiency. To increase the Fe content of rice, Goto et al.  transferred soybean ferritin gene into the plant. Using the rice seed storage protein glutelin promoter (GluB-1), they could target the expression of ferritin in developing seeds. The Fe content in transformed seeds was threefold compared to that in control seeds.
14.6 GENETIC MANIPULATION OF ORGANIC ACID BIOSYNTHESIS
It has been proposed that metal tolerance could be based on the organic acid-formed complexes. Ernst  observed high malate concentrations in Zn- and Cu-tolerant plants; also the content of citrate was increased. Hyperaccumulators are heavily loaded with these acids and acid anions might have some function in metal storage or plant internal metal transport. Free histidine has been found as a metal chelator in xylem exudates in plants that accumulate Ni and the amount of free histidine increases in Ni exposure . By modifying histidine metabolism, it might be possible to increase the Ni-accumulating capacity of plants.
During the past few years, several metal transporters have been isolated from Arabidopsis: Zn transporters ZIP1, 3, 4 ; Fe transporter IRT1 ; and Cu transporter COPT1 [126, 127]. Several transporters, like ZIP1, ZIP3 and IRT1, are expressed in response to metal deficiency. IRT1 may also play a role in the uptake of other metals because Cd, Zn, Co, and Mn inhibited Fe uptake of IRT1 . Changing the regulation of the expression of these transporters may modify the uptake of metals to the cells or organelles in a useful way.
Most of the studies aimed at determining the role of organic acid excretion have been carried out by comparing different plant species or nonisogenic lines of the same plant species. Plant transformation allows the production of genetically identical plants that differ only in one or a few genes. Taking advantage of this technology, the author's research group produced transgenic plants with an enhanced capacity to synthesize and excrete citrate. It was reasoned that, by overproducing citrate (one of the most powerful cation chelators in the organic acid group), the actual relevance of organic acids in several aspects of the plant-soil relationship could be elucidated.
To produce citrate-overproducing (CSb) plants, the coding sequence of the bacterial citrate synthase gene was placed under control of the 35S CaMV promoter and the nopaline synthase 3% end sequence (35S-CSb). This construct was used to transform tobacco and papaya plants. To determine whether the expression of a citrate synthase in plant cells leads to an increase in their citrate content, total and root extracts of transgenic lines were examined by HPLC and compared to control plants. It was found that the tobacco lines expressing the 35S-CSb construct had up to tenfold higher levels of citrate in their root tissue. The amount of citrate exuded by the roots of these transgenic lines was also increased up to fourfold as compared to control plants .
De la Fuente et al.  characterized the novel transgenic CSb lines to determine whether these plants were tolerant to aluminum. Experimental evidence suggests that the citrate-overproducing plants could tolerate a tenfold higher Al concentration than control plants. A mitochondrial citrate synthase of Arabidopsis thaliana was introduced into carrot (Daucus carota) cells by Agrobacterium tumefaciens. Several transgenic carrot cell lines that produced the Arabidopsis CS polypeptides and had high CS activity were identified. The increase in CS expression resulted in an enhanced capacity of phosphate uptake from insoluble sources of P in these transgenic cells [81,88].
More recently, this research group has shown that transgenic Arabidopsis plants that express high levels of the carrot citrate synthase have an enhanced aluminum tolerance. It has been reported that organic acid excretion by lupin plants constitutes a drain of 5 to 25% of the total fixed C; however, this does not appear to affect dry matter production significantly. This fact has also been confirmed in transgenic tobacco plants that overproduce citrate, which grow efficiently even at high levels of P-fertilization . The insights obtained from transgenic models highlight the potential of organic acid manipulation to generate novel crops more efficient in the use of soil P and well adapted for growth in marginal soils .
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