Root morphology and seed and leaf ionomic traits in a Brassica napus L. diversity panel show wide phenotypic variation and are characteristic of crop habit.

BACKGROUND: Mineral nutrient uptake and utilisation by plants are controlled by many traits relating to root morphology, ion transport, sequestration and translocation. The aims of this study were to determine the phenotypic diversity in root morphology and leaf and seed mineral composition of a polyploid crop species, Brassica napus L., and how these traits relate to crop habit. Traits were quantified in a diversity panel of up to 387 genotypes: 163 winter, 127 spring, and seven semiwinter oilseed rape (OSR) habits, 35 swede, 15 winter fodder, and 40 exotic/unspecified habits. Root traits of 14 d old seedlings were measured in a 'pouch and wick' system (n = ~24 replicates per genotype). The mineral composition of 3-6 rosette-stage leaves, and mature seeds, was determined on compost-grown plants from a designed experiment (n = 5) by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Seed size explained a large proportion of the variation in root length. Winter OSR and fodder habits had longer primary and lateral roots than spring OSR habits, with generally lower mineral concentrations. A comparison of the ratios of elements in leaf and seed parts revealed differences in translocation processes between crop habits, including those likely to be associated with crop-selection for OSR seeds with lower sulphur-containing glucosinolates. Combining root, leaf and seed traits in a discriminant analysis provided the most accurate characterisation of crop habit, illustrating the interdependence of plant tissues. CONCLUSIONS: High-throughput morphological and composition phenotyping reveals complex interrelationships between mineral acquisition and accumulation linked to genetic control within and between crop types (habits) in B. napus. Despite its recent genetic ancestry (<10 ky), root morphology, and leaf and seed composition traits could potentially be used in crop improvement, if suitable markers can be identified and if these correspond with suitable agronomy and quality traits.

Arabidopsis thaliana Yellow Stripe1-Like4 and Yellow Stripe1-Like6 localize to internal cellular membranes and are involved in metal ion homeostasis.

Several members of the Yellow Stripe1-Like (YSL) family of transporter proteins are able to transport metal-nicotianamine (NA) complexes. Substantial progress has been made in understanding the roles of the Arabidopsis YSLs that are most closely related to the founding member of the family, ZmYS1 (e.g., AtYSL1, AtYSL2 and AtYSL3), but there is little information concerning members of the other two well-conserved YSL clades. Here, we provide evidence that AtYSL4 and AtYSL6, which are the only genes in Arabidopsis belong to YSL Group II, are localized to vacuole membranes and to internal membranes resembling endoplasmic reticulum. Both single and double mutants for YSL4 and YSL6 were rigorously analyzed, and have surprisingly mild phenotypes, in spite of the strong and wide-ranging expression of YSL6. However, in the presence of toxic levels of Mn and Ni, plants with mutations in YSL4 and YSL6 and plants overexpressing GFP-tagged YSL6 showed growth defects, indicating a role for these transporters in heavy metal stress responses.

Selenium uptake, translocation, assimilation and metabolic fate in plants.

The chemical and physical resemblance between selenium (Se) and sulfur (S) establishes that both these elements share common metabolic pathways in plants. The presence of isologous Se and S compounds indicates that these elements compete in biochemical processes that affect uptake, translocation and assimilation throughout plant development. Yet, minor but crucial differences in reactivity and other metabolic interactions infer that some biochemical processes involving Se may be excluded from those relating to S. This review examines the current understanding of physiological and biochemical relationships between S and Se metabolism by highlighting their similarities and differences in relation to uptake, transport and assimilation pathways as observed in Se hyperaccumulator and non-accumulator plant species. The exploitation of genetic resources used in bioengineering strategies of plants is illuminating the function of sulfate transporters and key enzymes of the S assimilatory pathway in relation to Se accumulation and final metabolic fate. These strategies are providing the basic framework by which to resolve questions relating to the essentiality of Se in plants and the mechanisms utilized by Se hyperaccumulators to circumvent toxicity. In addition, such approaches may assist in the future application of genetically engineered Se accumulating plants for environmental renewal and human health objectives.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense.

The ability of Thlaspi goesingense to hyperaccumulate Ni seems to be governed in part by enhanced accumulation of Ni within leaf vacuoles. We have characterized genes from T. goesingense encoding putative vacuolar metal ion transport proteins, termed metal tolerance proteins (TgMTPs). These proteins contain all of the features of cation-efflux family members, and evidence indicates they are derived from a single genomic sequence (TgMTP1) that gives rise to an unspliced (TgMTP1t1) and a spliced (TgMTP1t2) transcript. Heterologous expression of these transcripts in yeast lacking the TgMTP1 orthologues COT1 and ZRC1 complements the metal sensitivity of these yeast strains, suggesting that TgMTP1s are able to transport metal ions into the yeast vacuole in a manner similar to COT1 and ZRC1. The unspliced and spliced TgMTP1 variants differ within a histidine-rich putative metal-binding domain, and these sequence differences are reflected as alterations in the metal specificities of these metal ion transporters. When expressed in yeast, TgMTP1t1 confers the highest level of tolerance to Cd, Co, and Zn, whereas TgMTP1t2 confers the highest tolerance to Ni. TgMTP1 transcripts are highly expressed in T. goesingense compared with orthologues in the nonaccumulators Arabidopsis thaliana, Thlaspi arvense, and Brassica juncea. We propose that the high-level expression of TgMTP1 in T. goesingense accounts for the enhanced ability of this hyperaccumulator to accumulate metal ions within shoot vacuoles.

Quantitative, chemically specific imaging of selenium transformation in plants.

Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-microm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 microM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3-0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2-0.3 compared with 3-4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 x 100 microm(2) regions of the same tissues.

Reduction and coordination of arsenic in Indian mustard.

The bioaccumulation of arsenic by plants may provide a means of removing this element from contaminated soils and waters. However, to optimize this process it is important to understand the biological mechanisms involved. Using a combination of techniques, including x-ray absorption spectroscopy, we have established the biochemical fate of arsenic taken up by Indian mustard (Brassica juncea). After arsenate uptake by the roots, possibly via the phosphate transport mechanism, a small fraction is exported to the shoot via the xylem as the oxyanions arsenate and arsenite. Once in the shoot, the arsenic is stored as an As(III)-tris-thiolate complex. The majority of the arsenic remains in the roots as an As(III)-tris-thiolate complex, which is indistinguishable from that found in the shoots and from As(III)-tris-glutathione. The thiolate donors are thus probably either glutathione or phytochelatins. The addition of the dithiol arsenic chelator dimercaptosuccinate to the hydroponic culture medium caused a 5-fold-increased arsenic level in the leaves, although the total arsenic accumulation was only marginally increased. This suggests that the addition of dimercaptosuccinate to arsenic-contaminated soils may provide a way to promote arsenic bioaccumulation in plant shoots, a process that will be essential for the development of an efficient phytoremediation strategy for this element.

Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species.

The ability of Thlaspi goesingense Hálácsy to hyperaccumulate Ni appears to be governed by its extraordinary degree of Ni tolerance. However, the physiological basis of this tolerance mechanism is unknown. We have investigated the role of vacuolar compartmentalization and chelation in this Ni tolerance. A direct comparison of Ni contents of vacuoles from leaves of T. goesingense and from the non-tolerant non-accumulator Thlaspi arvense L. showed that the hyperaccumulator accumulates approximately 2-fold more Ni in the vacuole than the non-accumulator under Ni exposure conditions that were non-toxic to both species. Using x-ray absorption spectroscopy we have been able to determine the likely identity of the compounds involved in chelating Ni within the leaf tissues of the hyperaccumulator and non-accumulator. This revealed that the majority of leaf Ni in the hyperaccumulator was associated with the cell wall, with the remaining Ni being associated with citrate and His, which we interpret as being localized primarily in the vacuolar and cytoplasm, respectively. This distribution of Ni was remarkably similar to that obtained by cell fractionation, supporting the hypothesis that in the hyperaccumulator, intracellular Ni is predominantly localized in the vacuole as a Ni-organic acid complex.

Molecular dissection of the role of histidine in nickel hyperaccumulation in Thlaspi goesingense (Hálácsy).

To understand the role of free histidine (His) in Ni hyperaccumulation in Thlaspi goesingense, we investigated the regulation of His biosynthesis at both the molecular and biochemical levels. Three T. goesingense cDNAs encoding the following His biosynthetic enzymes, ATP phosphoribosyltransferase (THG1, GenBank accession no. AF003347), imidazoleglycerol phosphate dehydratase (THB1, GenBank accession no. AF023140), and histidinol dehydrogenase (THD1, GenBank accession no. AF023141) were isolated by functional complementation of Escherichia coli His auxotrophs. Northern analysis of THG1, THD1, and THB1 gene expression revealed that each gene is expressed in both roots and shoots, but at the concentrations and dosage times of Ni treatment used in this study, these genes failed to show any regulation by Ni. We were also unable to observe any increases in the concentration of free His in root, shoot, or xylem sap of T. goesingense in response to Ni exposure. X-ray absorption spectroscopy of root and shoot tissue from T. goesingense and the non-accumulator species Thlaspi arvense revealed no major differences in the coordination of Ni by His in these tissues. We therefore conclude that the Ni hyperaccumulation phenotype in T. goesingense is not determined by the overproduction of His in response to Ni.

X-ray absorption spectroscopy of cadmium phytochelatin and model systems.

Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides. We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly. We have used the synthetic ions [Cd(SPh)4]2-, [Cd4(SPh)10]2- and [S4Cd10(SPh)16]4-as crystallographically defined models for the cadmium site. The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes. In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species. The Cd-S distance in the phytochelatin complex is 2.54 A. The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation. Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS. The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for [S4Cd10(SPh)16]4-. This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Isolation and partial characterization of a lead-accumulating Brassica juncea mutant.

A new screening method for non-destructive, high-sensitivity, high-throughput isolation of plant mutants capable of accumulating large amounts of heavy metals has been developed. This method is based on incubating seedlings in a solution containing radioisotopes of the metals of interest and visualizing the tissue accumulation of these metals with a phosphorimager. We used this technique to isolate mutants of Brassica juncea (L.) Czern with increased accumulation of Cd and Pb for use in phytoremediation, an emerging technology using plants to remediate polluted soil and water. Approximately 50,000 M2 seedlings were screened and 21 mutants were recovered that retained increased accumulation through the third generation. Mutant 7/15-1 is characterized by enhanced Pb accumulation per unit of root fresh weight, stunted root growth, and decreased root cell size. Data indicate that roots of 7/15-1 contain more cell-wall material on a fresh-weight basis than roots of the wild-type, which may at least partially explain its ability to accumulate more Pb.

PHYTOREMEDIATION.

Contaminated soils and waters pose a major environmental and human health problem, which may be partially solved by the emerging phytoremediation technology. This cost-effective plant-based approach to remediation takes advantage of the remarkable ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues. Toxic heavy metals and organic pollutants are the major targets for phytoremediation. In recent years, knowledge of the physiological and molecular mechanisms of phytoremediation began to emerge together with biological and engineering strategies designed to optimize and improve phytoremediation. In addition, several field trials confirmed the feasibility of using plants for environmental cleanup. This review concentrates on the most developed subsets of phytoremediation technology and on the biological mechanisms that make phytoremediation work.

The Role of Metal Transport and Tolerance in Nickel Hyperaccumulation by Thlaspi goesingense Halacsy.

Metal hyperaccumulators are plants that are capable of extracting metals from the soil and accumulating them to extraordinary concentrations in aboveground tissues (greater than 0.1% dry biomass Ni or Co or greater than 1% dry biomass Zn or Mn). Approximately 400 hyperaccumulator species have been identified, according to the analysis of field-collected specimens. Metal hyperaccumulators are interesting model organisms to study for the development of a phytoremediation technology, the use of plants to remove pollutant metals from soils. However, little is known about the molecular, biochemical, and physiological processes that result in the hyperaccumulator phenotype. We investigated the role of Ni tolerance and transport in Ni hyperaccumulation by Thlaspi goesingense, using plant biomass production, evapotranspiration, and protoplast viability assays, and by following short- and long-term uptake of Ni into roots and shoots. As long as both species (T. goesingense and Thlaspi arvense) were unaffected by Ni toxicity, the rates of Ni translocation from roots to shoots were the same in both the hyper- and nonaccumulator species. Our data suggest that Ni tolerance is sufficient to explain the Ni hyperaccumulator phenotype observed in hydroponically cultured T. goesingense when compared with the Ni-sensitive nonhyperaccumulator T. arvense.

Mechanisms of Cadmium Mobility and Accumulation in Indian Mustard.

Indian mustard (Brassica juncea L.), a high biomass crop plant, accumulated substantial amounts of cadmium, with bioaccumulation coefficients (concentration of Cd in dry plant tissue/concentration in solution) of up to 1100 in shoots and 6700 in roots at nonphytotoxic concentrations of Cd (0.1 [mu]g/mL) in solution. This was associated with a rapid accumulation of phytochelatins in the root, where the majority of the Cd was coordinated with sulfur ligands, probably as a Cd-S4 complex, as demonstrated by x-ray absorption spectroscopy. In contrast, Cd moving in the xylem sap was coordinated predominantly with oxygen or nitrogen ligands. Cd concentrations in the xylem sap and the rate of Cd accumulation in the leaves displayed similar saturation kinetics, suggesting that the process of Cd transport from solution through the root and into the xylem is mediated by a saturable transport system(s). However, Cd translocation to the shoot appeared to be driven by transpiration, since ABA dramatically reduced Cd accumulation in leaves. Within leaves, Cd was preferentially accumulated in trichomes on the leaf surface, and this may be a possible detoxification mechanism.

Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants.

Toxic metal pollution of waters and soils is a major environmental problem, and most conventional remediation approaches do not provide acceptable solutions. The use of specially selected and engineered metal-accumulating plants for environmental clean-up is an emerging technology called phytoremediation. Three subsets of this technology are applicable to toxic metal remediation: (1) Phytoextraction--the use of metal-accumulating plants to remove toxic metals from soil; (2) Rhizofiltration--the use of plant roots to remove toxic metals from polluted waters; and (3) Phytostabilization--the use of plants to eliminate the bioavailability of toxic metals in soils. Biological mechanisms of toxic metal uptake, translocation and resistance as well as strategies for improving phytoremediation are also discussed.

MgATP-Dependent Transport of Phytochelatins Across the Tonoplast of Oat Roots.

In Cd-exposed oat (Avena sativa) roots Cd was found to be associated primarily with the phytochelatin ([gamma]-glutamylcysteinyl)3-glutamic acid [([gamma]EC)3G], with a peptide to Cd ratio of 1:3 (cysteine to Cd ratio of 1:1), even though both ([gamma]EC)2G and ([gamma]EC)3G were present in the roots. Phytochelatins are known to accumulate in the vacuoles of plant cells on exposure to Cd, but the mechanism is not clear. Here we present evidence for the transport of the phytochelatins ([gamma]EC)3G and ([gamma]EC)2G as well as the Cd complex Cd-([gamma]EC)3G across the tonoplast of oat roots. Transport of ([gamma]EC)3G had a Km, for MgATP of 0.18 mM and a Vmax of 0.7 to 1 nmol mg-1 protein min-1. Transport of ([gamma]EC)3G was also energized by MgGTP and to a lesser extent MgUTP and was highly sensitive to orthovanadate, with a 50%-inhibitory concentration of 0.9 [mu]M. The Cd complex Cd-([gamma]EC)3G and ([gamma]EC)2G were also transported in a MgATP-dependent, vanadate-sensitive manner. Therefore, this process is a candidate for the transport of both phytochelatins, and Cd as its peptide complex, from the cytoplasm into the vacuole.