Constitutive high-level SOS1 expression and absence of HKT1;1 expression in the salt-accumulating halophyte Salicornia dolichostachya.

We investigated the effects of salinity on ion accumulation and expression of candidate salt tolerance genes in the highly tolerant salt accumulating halophyte Salicornia dolichostachya and the taxonomically related glycophytic Spinacia oleracea. S. dolichostachya, in comparison with S. oleracea, constitutively expressed SOS1 at a high level, but did not detectably express HKT1;1. These findings suggest that the constitutive high level of shoot salt accumulation in S. dolichostachya is accomplished through enhancement of SOS1-mediated Na(+) xylem loading, in combination with complete suppression of HKT1;1-mediated Na(+) retrieval from the xylem. Our findings demonstrate the importance of gene expression comparisons between highly tolerant halophytes and taxonomically related glycophytes to improve the understanding of mechanisms of Na(+) movement and salt tolerance in plants.

Plant metallothioneins--metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F(3) families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F(3) sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

Variation in root-to-shoot translocation of cadmium and zinc among different accessions of the hyperaccumulators Thlaspi caerulescens and Thlaspi praecox.

Efficient root-to-shoot translocation is a key trait of the zinc/cadmium hyperaccumulators Thlaspi caerulescens and Thlaspi praecox, but the extent of variation among different accessions and the underlying mechanisms remain unclear. Root-to-shoot translocation of Cd and Zn and apoplastic bypass flow were determined in 10 accessions of T. caerulescens and one of T. praecox, using radiolabels (109)Cd and (65)Zn. Two contrasting accessions (Pr and Ga) of T. caerulescens were further characterized for TcHMA4 expression and metal compartmentation in roots. Root-to-shoot translocation of (109)Cd and (65)Zn after 1 d exposure varied 4.4 to 5-fold among the 11 accessions, with a significant correlation between the two metals, but no significant correlation with uptake or the apoplastic bypass flow. The F(2) progeny from a cross between accessions from Prayon, Belgium (Pr) and Ganges, France (Ga) showed a continuous phenotype pattern and transgression. There was no significant difference in the TcHMA4 expression in roots between Pr and Ga. Compartmentation analysis showed a higher percentage of (109)Cd sequestered in the root vacuoles of Ga than Pr, the former being less efficient in translocation than the latter. Substantial natural variation exists in the root-to-shoot translocation of Cd and Zn, and root vacuolar sequestration may be an important factor related to this variation.

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens.

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.

QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens.

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).

Co-segregation analysis of cadmium and zinc accumulation in Thlaspi caerulescens interecotypic crosses.

• Cadmium (Cd) hyperaccumulation in Thlaspi caerulescens varies among ecotypes. Here we investigated segregation of Cd and zinc (Zn) accumulation in F2 crosses between high (Ganges) and low (Prayon) Cd-accumulating ecotypes. • Accumulation was measured in plants grown in compost treated with 5 and 100 mg kg-1 Cd and Zn, respectively, and in hydroponics with 50 m Zn and 10 or 50 m Cd. Another hydroponic experiment examined the relationship between Cd tolerance and accumulation. • Parental phenotype distributions for shoot metal concentrations were distinct for Cd, but not consistent for Zn. Shoot Cd and Zn in F2 s varied continuously, with significant transgression for Zn in all treatments. Shoot Cd correlated strongly with shoot manganese (Mn), and to a lesser degree with shoot Zn. Shoot Cd concentrations in the Cd nontolerant F2 s were lower than, or similar to, those in the Cd-tolerant F2 s. • We conclude that Cd and Zn accumulation is governed by multiple genes, and that Cd tolerance and accumulation are independent traits in T. caerulescens. Two uptake systems with distinctive affinities for Cd, Zn and Mn are proposed.

The role of phytochelatins in arsenic tolerance in the hyperaccumulator Pteris vittata.

• Pteris vittata was the first identified arsenic (As) hyperaccumulator. Here we investigated whether phytochelatins (PCs) are involved in the hypertolerance of arsenic by P. vittata. • P. vittata was exposed to 0-500 µm arsenate for 5 d, or to 50 µm arsenate for 0-7 d. In addition, l-buthionine-sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, was used in combination with different arsenate exposures. The relationships between As accumulation and the concentrations of PCs and glutathione (GSH) were examined. • PC synthesis was induced upon exposure to arsenate in P. vittata, with only PC2 detected in the plant. The As concentration correlated significantly with PC2 concentration in both roots and shoots, but not with GSH. The molar ratio of PC-SH to As was c. 0.09 and 0.03 for shoots and roots, respectively, suggesting that only a small proportion (1-3%) of the As in P. vittata can be complexed with PCs. In the presence of arsenate, addition of BSO decreased PC2 concentrations in roots and shoots by 89-96% and 30-33%, respectively. BSO alone was found to inhibit root growth of P. vittata markedly. • The results suggest that PCs play a limited role in the hypertolerance of As in P. vittata.

Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression. Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Phytochelatins are involved in differential arsenate tolerance in Holcus lanatus.

Arsenate tolerance is conferred by suppression of the high-affinity phosphate/arsenate uptake system, which greatly reduces arsenate influx in a number of higher plant species. Despite this suppressed uptake, arsenate-tolerant plants can still accumulate high levels of As over their lifetime, suggesting that constitutive detoxification mechanisms may be required. Phytochelatins are thiol-rich peptides, whose production is induced by a range of metals and metalloids including arsenate. This study provides evidence for the role of phytochelatins in the detoxification of arsenate in arsenate-tolerant Holcus lanatus. Elevated levels of phytochelatin were measured in plants with a range of tolerance to arsenate at equivalent levels of arsenate stress, measured as inhibition of root growth. The results suggest that arsenate tolerance in H. lanatus requires both adaptive suppression of the high-affinity phosphate uptake system and constitutive phytochelatin production.

Derivatization of phytochelatins from Silene vulgaris, induced upon exposure to arsenate and cadmium: comparison of derivatization with Ellman's reagent and monobromobimane.

Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)()-Gly, with n = 2-11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5, 5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)(3)-As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2-4 (PC(2)(-)(4)) are compared. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC(2) to 27.4% and 50.2% for PC(4). This decrease is most likely due to steric hindrance. Correction of measured thiol concentration is therefore advised for better quantification of PC concentrations in plant material.

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.

Properties of enhanced tonoplast zinc transport in naturally selected zinc-tolerant silene vulgaris

It was demonstrated recently that isolated tonoplast vesicles derived from plants of a Zn-tolerant ecotype of Silene vulgaris accumulate more Zn than vesicles derived from a Zn-sensitive ecotype. We have now characterized the tonoplast-transport system that causes this uptake difference and demonstrated its genetic correlation to Zn tolerance using plant crosses. We conclude that the tonoplast Zn uptake system of the tolerant ecotype differs greatly in its characteristics from that of the sensitive one, with the most prominent differences being its insensitivity to protonophores and ortho-vanadate and its stimulation by Mg-GTP. These differences in characteristics are most likely due to the fact that Zn can be taken up by two or more parallel pathways, which are not present in the same proportions in both ecotypes. In both ecotypes, Zn is actively transported across the tonoplast (temperature coefficient > 1.6), most likely as a free ion, since citrate does not accumulate in vesicles. Most importantly, the uptake difference found using the ecotypes was also found between homozygous Zn-tolerant and Zn-sensitive F3 plants, proving the genetic correlation between increased tonoplast Zn transport and naturally selected Zn tolerance in S. vulgaris.

Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation.

We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.

Differential tolerance to copper and zinc of micropropagated birches tested in hydroponics.

Copper and zinc tolerances of 10 micropropagated birch (Betula pendula and B. pubescens) clones were studied in hydroponic culture. Tolerance indices were determined, based on the mean growth rate of the longest root in 1 wk. A seed-derived clone (142A), from a lead/Zn-contaminated site showed more tolerance to Cu and Zn than bud-derived clones (HA02 and HA18) from a Cu/nickel-contaminated site or an ozone-tolerant clone (KL-2-M) from an uncontaminated area. For Cu, the EC50 values were 30, 14, 8 and 11 µM in clones 142A, HA02, HA18 and KL-2-M, respectively. FOT Zn, the EC50 s were 4000 and 350 µM in clones 142A and KL-2-M, respectively. The relative Cu and Zn tolerances of the other clones were estimated by growing the plants in 30 µ CuSO4 , and in 2000 or 350 µM ZnSO4 , respectively. It is of interest that the Zn-tolerant clone 142A was tolerant to Cu, although this metal was present at a very low concentration in the soil where the parent tree grows. Another clone (142B), from another seed of the same parent tree, was tolerant neither to Zn nor Cu. Compared with their own EC20 s for root growth for Cu, 142A took up more Cu than KL-2-M, suggesting that the higher tolerance of the former clone is not explained by reduced uptake of Cu. The Zn uptake in clones 142A and KL-2-M was studied at 4000 µM and 800 µM Zn, respectively. Interestingly, the roots of both clones contained the same amount of Zn, even though clone 142A was exposed to a fivefold concentration of Zn.

Phytochelatins in Cadmium-Sensitive and Cadmium-Tolerant Silene vulgaris (Chain Length Distribution and Sulfide Incorporation).

In response to a range of Cd concentrations, the root tips of Cd-tolerant plants of Silene vulgaris exhibit a lower rate of PC production accompanied by a lower rate of longer chain PC synthesis than those of Cd-sensitive plants. At the same Cd exposure level, stable PC-Cd complexes are more rapidly formed in the roots of Cd-sensitive plants than in those of tolerant plants. At an equal PC concentration in the roots, the PC composition and the amount of sulfide incorporated per unit of PC-thiol is the same in both populations. Although these compounds might play some role in mechanisms that contribute to Cd detoxification, the ability to produce these compounds in greater amounts is not, itself, the mechanism that produces increased Cd tolerance in tolerant S. vulgaris plants.

Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain?

Plants from nontolerant and copper-tolerant populations of Silene vulgaris both produce phytochelatins upon exposure to copper. The threshold copper concentration for induction of phytochelatin and the copper concentration at which maximum phytochelatin contents occurs increase proportionally with the level of tolerance to copper. When exposed to their own highest no-effect concentration or 50%-effect concentration of copper for root growth, tolerant and nontolerant plants exhibit equal phytochelatin contents in the root apex, which is the primary copper target. This also holds for distinctly tolerant nonsegregating F(3) families, derived from a single cross of a nontolerant plant to a tolerant one. Therefore, the phytochelatin content of the root apex can be used as a quantitative tolerance-independent measure of the degree of toxicity experienced by the plant. Differential copper tolerance in S. vulgaris does not appear to rely on differential phytochelatin production.

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus.

The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.