Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.     

The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes

Using the gamma-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulphoximine (BSO), the role for phytochelatins (PCs) was evaluated in Cu, Cd, Zn, As, Ni, and Co tolerance in non-metallicolous and metallicolous, hypertolerant populations of Silene vulgaris (Moench) Garcke, Thlaspi caerulescens J.&C. Presl., Holcus lanatus L., and Agrostis castellana Boiss. et Reuter. Based on plant-internal PC-thiol to metal molar ratios, the metals' tendency to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in non-metallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were consistently unaffected by BSO treatment, both in non-metallicolous and hypertolerant plants, suggesting that PC-based sequestration is not essential for constitutive tolerance or hypertolerance to these metals. Cd sensitivity was considerably increased by BSO, though exclusively in plants lacking Cd hypertolerance, suggesting that adaptive cadmium hypertolerance is not dependent on PC-mediated sequestration. BSO dramatically increased As sensitivity, both in non-adapted and As-hypertolerant plants, showing that PC-based sequestration is essential for both normal constitutive tolerance and adaptive hypertolerance to this metalloid. The primary function of PC synthase in plants and algae remains elusive.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

Synthesis of low molecular weight thiols in response to Cd exposure in Thlaspi caerulescens

In this study, we investigated the accumulation of phytochelatins (PCs) and other low molecular weight (LMW) thiols in response to Cd exposure in two contrasting ecotypes differing in Cd accumulation. Using a root elongation test, we found that the highly accumulating ecotype Ganges was more tolerant to Cd than the low Cd-accumulation ecotype Prayon. l -buthionine-(S,R)-sulphoximine (BSO), a potent inhibitor of the γ -glutamylcysteine synthetase ( γ -ECS) (an enzyme involved in the PC biosynthetic pathway), increased the Cd sensitivity of Prayon, but had no effect on Ganges. Although PC accumulation increased in response to Cd exposure, no significant differences were observed between the two ecotypes. Cd exposure induced a dose-dependent accumulation of both Cys and a still unidentified LMW thiol in roots of both ecotypes. Root accumulation of Cys and this thiol was higher in Ganges than in Prayon; the ecotypic differences were more pronounced when the plants were treated with BSO. These findings suggest that PCs do not contribute to the Cd hypertolerance displayed by the Ganges ecotype of Thlaspi caerulescens , whereas Cys and other LMW thiols might be involved.     

Arabidopsis thaliana, a new tool to investigate Polymyxa betae-host interactions

Little is known about the genome of Polymyxa betae and its interactions with sugar beet, due partly to the obligate nature of the protist and the patents on Beta vulgaris sequences. The identification of an ecotype of Arabidopsis thaliana compatible with the protist would help to improve this knowledge. The infection and development of P. betae in 14 worldwide ecotypes of A. thaliana were studied. The detection of plasmodia and resting spores and the production of zoospores in the roots of A. thaliana were obtained in three bioassays, using automatic immersion systems and individual glass tubes. Detection was done using molecular detection and microscopy. Compatible interactions were established between 13 A. thaliana ecotypes of the 14 that were tested and the monosporosoric Belgian strain of P. betae, A26-41. The ecotype Cvi-0 (N1096), from the Cape Verde Islands, was the most compatible with the protist. This ecotype is also susceptible to Plasmodiophora brassicae, another plasmodiophorid. Polymyxa betae infection in A. thaliana was relatively very low compared with B. vulgaris, but every stage of the life cycle of the protist was present. The spore-forming phase was promoted at the expense of the sporangial phase, probably caused by the stress of this new environment. In addition, the protist revealed a new phenotype. This new model study will allow molecular tools available for A. thaliana to be used in order to gain a better understanding of the P. betae-plant interaction during the spore-forming phase.     

Mitochondrial DNA variations and nuclear RFLPs reflect different genetic similarities among 23 Arabidopsis thaliana ecotypes

The mitochondrial genome of 23 Arabidopsis thaliana ecotypes was analysed by Southern hybridization in total cellular DNA. Firstly, the extent of divergence between the mitochondrial genomes in closely related lines of one plant species and secondly, the use of mitochondrial versus nuclear RFLPs to determine evolutionary relationships between Arabidopsis ecotype isolates was investigated. Highly divergent stoichiometries of alternative mitochondrial genome arrangements characterize individual ecotypes including the complete loss of a 5 kb region from ecotype Landsberg without apparent effect on plant viability. The genetic similarities between ecotypes suggested by mitochondrial genome arrangements differ from those deduced from 18 nuclear RFLP loci (CAPS markers). Similarity of nuclear RFLP patterns among the 23 Arabidopsis ecotypes neitehr correlates with their geographic origin nor with the observed mitochondrial genome arrangements. A promiscuous mitochondrial sequence insertion previously identified in ecotype Columbia is also found in the nuclear genomes of ecotypes Eifel, Enkheim and Hilversum. Two ecotypes (Eifel and Tabor) displaying identical RFLP patterns at all 18 nuclear loci show differences in both this sequence transfer and a mitochondrial DNA recombination event.     

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.     

Susceptibility and symptom development in Arabidopsis thaliana to Tobacco mosaic virus is influenced by virus cell-to-cell movement

To identify host factors that regulate susceptibility to Tobacco mosaic virus (TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.     

Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.

The responses of a collection of Arabidopsis thaliana ecotypes to mechanical inoculation with turnip mosaic potyvirus were assessed. The virus induced characteristic severe symptoms of infection in systemically infected plants. Resistance was found in four ecotypes: Bay-0, Di-0, Er-0, and Or-0. Enzyme-linked immunosorbent assay results of the resistant ecotypes suggested that ecotypes Di-0, Er-0, and Or-0 actually consist of mixed genotypes with resistances acting at different levels in the virus life cycle. Another form of resistance was found in ecotype Bay-0, for which several lines of evidence indicated an interference with viral cell-to-cell movement in the inoculated leaves.     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.     

Metabolite profiling for plant functional genomics

Multiparallel analyses of mRNA and proteins are central to today's functional genomics initiatives. We describe here the use of metabolite profiling as a new tool for a comparative display of gene function. It has the potential not only to provide deeper insight into complex regulatory processes but also to determine phenotype directly. Using gas chromatography/mass spectrometry (GC/MS), we automatically quantified 326 distinct compounds from Arabidopsis thaliana leaf extracts. It was possible to assign a chemical structure to approximately half of these compounds. Comparison of four Arabidopsis genotypes (two homozygous ecotypes and a mutant of each ecotype) showed that each genotype possesses a distinct metabolic profile. Data mining tools such as principal component analysis enabled the assignment of "metabolic phenotypes" using these large data sets. The metabolic phenotypes of the two ecotypes were more divergent than were the metabolic phenotypes of the single-loci mutant and their parental ecotypes. These results demonstrate the use of metabolite profiling as a tool to significantly extend and enhance the power of existing functional genomics approaches.     

Characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens

Uptake of Cd and Zn by intact seedlings of two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens was characterized using radioactive tracers. Uptake of Cd and Zn at 2 degrees C was assumed to represent mainly apoplastic binding in the roots, whereas the difference in uptake between 22 degrees C and 2 degrees C represented metabolically dependent influx. There was no significant difference between the two ecotypes in the apoplastic binding of Cd or Zn. Metabolically dependent uptake of Cd was 4.5-fold higher in the high Cd-accumulating ecotype, Ganges, than in the low Cd-accumulating ecotype, Prayon. By contrast, there was only a 1.5-fold difference in the Zn uptake between the two ecotypes. For the Ganges ecotype, Cd uptake could be described by Michaelis-Menten kinetics with a V(max) of 143 nmol g(-1) root FW h(-1) and a K(m) of 0.45 microM. Uptake of Cd by the Ganges ecotype was not inhibited by La, Zn, Cu, Co, Mn, Ni or Fe(II), and neither by increasing the Ca concentration. By contrast, addition of La, Zn or Mn, or increasing the Ca concentration in the uptake solution decreased Cd uptake by Prayon. Uptake of Ca was larger in Prayon than in Ganges. The results suggest that Cd uptake by the low Cd-accumulating ecotype (Prayon) may be mediated partly via Ca channels or transporters for Zn and Mn. By contrast, there may exist a highly selective Cd transport system in the root cell membranes of the high Cd-accumulating ecotype (Ganges) of T. caerulescens.     

Arabidopsis PAI gene arrangements, cytosine methylation and expression.

Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression.     

Subcellular localization of cadmium in roots and leaves of Arabidopsis thaliana

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.     

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.