Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens

* In this study we address the impact of changes in plant heavy metal, (i.e. zinc (Zn) and cadmium (Cd)) status on metal accumulation in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. * Thlaspi caerulescens plants were grown hydroponically on both high and low Zn and Cd regimes and whole-shoot and -root metal accumulation, and root (109)Cd(2+) influx were determined. * High-Zn-grown (500 microm Zn) plants were found to be more Cd-tolerant than plants grown in standard Zn conditions (1 microm Zn). Furthermore, shoot Cd accumulation was significantly greater in the high-Zn-grown plants. A positive correlation was also found between shoot Zn accumulation and increased plant Cd status. Radiotracer (109)Cd root flux experiments demonstrated that high-Zn-grown plants maintained significantly higher root Cd(2+) influx than plants grown on 1 microm Zn. It was also found that both nickel (Ni) and copper (Cu) shoot accumulation were stimulated by high plant Zn status, while manganese (Mn) accumulation was not affected. * A speculative model is presented to explain these findings, suggesting that xylem loading may be one of the key sites responsible for the hyperaccumulation of Zn and Cd accumulation in Thlaspi caerulescens.     

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).     

Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.     

Assessment of plants from the Brassicaceae family as genetic models for the study of nickel and zinc hyperaccumulation

We report on the second phase of a programme to select a relative of Arabidopsis thaliana for use in large-scale molecular genetic studies of nickel (Ni) and zinc (Zn) hyperaccumulation. We also report on the relatedness among Thlaspi caerulescens accessions and the utility of using O-acetyl-L-serine as a marker for Ni and Zn hyperaccumulation potential. Twenty-seven new accessions of metal-accumulating species collected in the Czech Republic, France, Greece, Italy, Slovenia and the USA during Spring-Summer 2002 were evaluated. The criteria established for selection were hyperaccumulation of metals (Ni and Zn); compact growth habit; reasonable time to flowering; production of > or = 1000 seeds per plant; self-fertility; compact diploid genome; high sequence similarity to A. thaliana; > or = 0.1% transformation efficiency with easy selection. We conclude that the best candidate identified in the first phase was the best candidate overall: T. caerulescens accession St Félix de Pallières.     

Cadmium hyperaccumulation and reproductive traits in natural Thlaspi caerulescens populations

During the last decade, the metal hyperaccumulating plants have attracted considerable attention because of their potential use in decontamination of heavy metal contaminated soils. However, in most species, little is known regarding the function, the ecological and the evolutionary significances of hyperaccumulation. In our study, we investigated the parameters influencing the Cd concentration in plants as well as the biological implications of Cd hyperaccumulation in nine natural populations of Thlaspi caerulescens. First, we showed that Cd concentration in the plant was positively correlated with plant Zn, Fe, and Cu concentrations. This suggested that the physiological and/or molecular mechanisms for uptake, transport and/or accumulation of these four heavy metals interact with each other. Second, we specified a measure of Cd hyperaccumulation capacity by populations and showed that T. caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth, by developing more and bigger leaves, taller stems, and produced more fruits and heavier seeds. These results suggest a tolerance/disposal role of Cd hyperaccumulation in this species.     

Zinc in plants

Zinc (Zn) is an essential component of thousands of proteins in plants, although it is toxic in excess. In this review, the dominant fluxes of Zn in the soil-root-shoot continuum are described, including Zn inputs to soils, the plant availability of soluble Zn(2+) at the root surface, and plant uptake and accumulation of Zn. Knowledge of these fluxes can inform agronomic and genetic strategies to address the widespread problem of Zn-limited crop growth. Substantial within-species genetic variation in Zn composition is being used to alleviate human dietary Zn deficiencies through biofortification. Intriguingly, a meta-analysis of data from an extensive literature survey indicates that a small proportion of the genetic variation in shoot Zn concentration can be attributed to evolutionary processes whose effects manifest above the family level. Remarkable insights into the evolutionary potential of plants to respond to elevated soil Zn have recently been made through detailed anatomical, physiological, chemical, genetic and molecular characterizations of the brassicaceous Zn hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri.     

Zinc tolerance and hyperaccumulation in F1 and F2 offspring from intra and interecotype crosses of Thlaspi caerulescens

The relationship between zinc (Zn) tolerance and hyperaccumulation in Thlaspi caerulescens was investigated from F1 and F2 crosses within and among metallicolous and nonmetallicolous Mediterranean populations. F1 offspring were grown on increasingly Zn-enriched soils to test Zn enrichment effects, and many families of F2 offspring were grown on a Zn-rich soil. Tolerance of F1 offspring depended on stress intensity. Tolerance of interecotype crosses was intermediate between that of the intraecotype crosses. No difference in Zn accumulation appeared among the F1 offspring from the three crosses involving metallicolous parents. Otherwise, none of these offspring exceeded the Zn hyperaccumulation threshold (10,000 mg kg(-1)), unlike the nonmetallicolous ones. The latter also showed the highest mortality. In some F2 families from interecotype crosses, hyperaccumulation values exceeded 15,000 mg kg(-1) in nontolerant offspring, whereas tolerant offspring displayed lower values (c. 10,000 mg kg(-1)). There was no difference between tolerant and nontolerant offspring when they showed low hyperaccumulation. Therefore, the relationship between tolerance and hyperaccumulation in F1 and F2 crosses depended on the hyperaccumulation level of plants.     

A quantitative trait loci analysis of zinc hyperaccumulation in Arabidopsis halleri

The mechanisms of metal hyperaccumulation are still not understood, so we conducted a quantitative trait locus (QTL) analysis of zinc (Zn) hyperaccumulation in Arabidopsis halleri, in a cross between this and its sister species, A. petraea, in order to determine the number and approximate location of the genomic regions significantly contributing to this adaptation. An F2 cross between the two species was made, and the leaf Zn concentration of 92 individuals was measured at both low (10 microm) and high (100 microm) Zn concentrations. Twenty-five markers were established that were distributed on all of the eight chromosomes. Mapping of the markers established that they were essentially collinear with previous studies. QTLs exceeding a logarithm to the base 10 of the odds (LOD) value of 3 were found on chromosomes 4 (low Zn), 6 (high Zn) and 7 (both high and low Zn). Evidence for a QTL on chromosome 3 (low Zn) was also found. This analysis validates a previously used method of QTL analysis, based on microarray analysis of segregating families. Genes that have altered during the evolution of this character should also be QTL: this analysis calls into question a number of candidate genes from consideration as such primary genes because they do not appear to be associated with QTLs.     

Cadmium accumulation in populations of Thlaspi caerulescens and Thlaspi goesingense

The capacity to accumulate cadmium (Cd) and zinc (Zn) was compared in Thlaspi goesingense and four populations of Thlaspi caerulescens . Two populations of T. caerulescens were grown in hydroponics at five concentrations of Cd. In addition, plants were grown in pots containing compost in which three different concentrations of Cd and two concentrations of Zn were added. A field trial was conducted to compare Zn and Cd uptake by three populations of T. caerulescens on nine selected plots of the Woburn Market Garden Experiment (UK) which had been contaminated to different degrees with heavy metals owing to past applications of sewage sludge. Results show that the four populations of T. caerulescens had the same ability to hyperaccumulate Zn but were significantly different in terms of Cd accumulation. Two populations of T. caerulescens from Southern France accumulated much more Cd than the populations from Prayon (Belgium) and Whitesike (UK). Generally, uptake of Cd was not decreased by increased concentrations of Zn in the substrate. These results indicate that the mechanisms of Cd and Zn hyperaccumulation are not identical in this species. This is the first report of hyperaccumulation of Cd by T. goesingense , but the growth of this species was markedly reduced by the large concentrations of Zn in the substrate. Future work should focus on the differences between Cd and Zn uptake in hyperaccumulator plants at the species and population level.     

Construction of a linkage map based on a <Emphasis Type="Italic">Lathyrus sativus</Emphasis> backcross population and preliminary investigation of QTLs associated with resistance to ascochyta blight

A linkage map of the Lathyrus sativus genome was constructed using 92 backcross individuals derived from a cross between an accession resistant (ATC 80878) to ascochyta blight caused by Mycosphaerella pinodes and a susceptible accession (ATC 80407). A total of 64 markers were mapped on the backcross population, including 47 RAPD, seven sequence-tagged microsatellite site and 13 STS/CAPS markers. The map comprised nine linkage groups, covered a map distance of 803.1 cM, and the average spacing between markers was 15.8 cM. Quantitative trait loci (QTL) associated with ascochyta blight resistance were detected using single-point analysis and simple and composite interval mapping. The backcross population was evaluated for stem resistance in temperature-controlled growth room trials. One significant QTL, QTL1, was located on linkage group 1 and explained 12% of the phenotypic variation in the backcross population. A second suggestive QTL, QTL2, was detected on linkage group 2 and accounted for 9% of the trait variation. The L. sativus R-QTL regions detected may be targeted for future intergenus transfer of the trait into accessions of the closely related species Pisum sativum.     

Identification of quantitative trait loci influencing foliar concentrations of terpenes and formylated phloroglucinol compounds in Eucalyptus nitens

Leaves of eucalypt species contain a variety of plant secondary metabolites, including terpenoids and formylated phloroglucinol compounds (FPCs). Both terpene and FPC concentrations are quantitative traits that can show large variation within a population and have been shown to be heritable. The molecular genetic basis of this variation is currently unknown. Progeny from a field trial of a three-generation mapping pedigree of Eucalyptus nitens were assayed for terpenes and FPCs. Quantitative trait loci (QTL) analyses were conducted using a map constructed from 296 markers to locate regions of the genome influencing foliar concentrations of these plant secondary compounds. A large number of significant QTL for 14 traits were located across nine linkage groups, with significant clustering of QTL on linkage groups 7, 8 and 9. As expected, QTL for biosynthetically related compounds commonly colocated, but QTL for unrelated monterpenes and FPCs also mapped closely together. Colocation of these QTL with mapped candidate genes from the various biosynthetic pathways, and subsequent use of these genes in association mapping, will assist in determining the causes of variation in plant secondary metabolites in eucalypts.     

甘蓝型油菜遗传图谱的构建及单株产量构成因素的QTL分析

采用常规品系04-1139与高产多角果品系05-1054构建的F2代群体为作图群体, 运用SSR(Simple sequence repeat)和SRAP(Sequence-related amplified polymorphism)构建分子标记遗传图谱并对甘蓝型油菜单株产量构成因素进行QTL分析。遗传图谱包含200个分子标记, 分布于19个连锁群上, 总长度1 700.23 cM, 标记间的平均距离8.50 cM。采用复合区间作图法(Composite interval mapping, CIM)对单株产量构成因素(单株有效角果数、每果粒数和千粒重)进行QTL分析, 共检测到12个QTL: 其中单株有效角果数4个QTL, 分别解释表型变异为35.64%、12.96%、28.71%和34.02%; 每果粒数获得5个QTL, 分别解释表型变异为8.41%、7.87%、24.37%、8.57%和14.31%; 千粒重获得３个QTL, 分别解释表型变异为2.33%、1.81%和1.86%。结果表明: 同一性状的等位基因增效作用可以同时来自高值亲本和低值亲本; 文章中与主效QTL连锁的标记可用于油菜产量性状的分子标记辅助选择和聚合育种。     

Cadmium hyperaccumulation protects Thlaspi caerulescens from leaf feeding damage by thrips (Frankliniella occidentalis)

Metal hyperaccumulation has been proposed as a plant defensive strategy. Here, we investigated whether cadmium (Cd) hyperaccumulation protected Thlaspi caerulescens from leaf feeding damage by thrips (Frankliniella occidentalis). Two ecotypes differing in Cd accumulation, Ganges (high) and Prayon (low), were grown in compost amended with 0-1000 mg Cd kg(-1) in two experiments under glasshouse conditions. F2 and F3 plants from the Prayon x Ganges crosses were grown with 5 mg Cd kg(-1). Plants were naturally colonized by thrips and the leaf feeding damage index (LFDI) was assessed. The LFDI decreased significantly with increasing Cd in both ecotypes, and correlated with shoot Cd concentration in a log-linear fashion. Prayon was more attractive to thrips than Ganges, but the ecotypic difference in the LFDI was largely accounted for by the shoot Cd concentration. In the F2 and F3 plants, the LFDI correlated significantly and negatively with shoot Cd, but not with shoot zinc (Zn) or sulphur (S) concentrations. We conclude that Cd hyperaccumulation deters thrips from feeding on T. caerulescens leaves, which may offer an adaptive benefit to the plant.     

Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues

Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL) mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL) for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.     

Mapping quantitative trait loci associated with arsenic accumulation in rice (Oryza sativa)

The quantitative trait loci (QTLs) associated with arsenic (As) accumulation in rice were mapped using a doubled haploid population established by anther culture of F1 plants from a cross between a Japonica cultivar CJ06 and an Indica cultivar TN1 (Oryza sativa). Four QTLs for arsenic (As) concentrations were detected in the map. At the seedling stage, one QTL was mapped on chromosome 2 for As concentrations in shoots with 24.4% phenotypic variance and one QTL for As concentrations in roots was detected on chromosome 3. At maturity, two QTLs for As concentrations in grains were found on chromosomes 6 and 8, with 26.3 and 35.2% phenotypic variance, respectively. No common loci were detected among these three traits. Interestingly, the QTL on chromosome 8 was found to be colocated for As concentrations in grain at maturity and shoot phosphorus (P) concentrations at seedling stage. These results provide an insight into the genetic basis of As uptake and accumulation in rice, and will be useful in identifying genes associated with As accumulation.     

Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations

Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P  ≤ 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24–67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.     

Natural variation of nickel,zinc and cadmium (hyper)accumulation in facultative serpentinophytes Noccaea kovatsii and N. praecox

Aims

Data on the variability of hyperaccumulation potential of the facultative serpentinophytes Noccaea kovatsii and N. praecox on different geological substrates are scarce. The aim of this study was to assess the accumulation potential of these two species from ultramafic and non-ultramafic substrates, with special emphasis on the hyperaccumulation of Ni, Zn and Cd.

Methods

Samples of plants and corresponding soils were collected from 16 sites covering a wide range of geochemistry. Elemental concentrations were determined in the roots, shoots and the rhizosphere soils. The pH, particle size distribution and content of organic matter were also determined in the soil samples.

Results

All ultramafic accessions of both species hyperaccumulated Ni with high intraspecific variability. Only one accession of N. kovatsii from a schist soil hyperaccumulated Zn, with also a high Cd accumulation. Accumulation and translocation of Ni in both species were much higher in the ultramafic accessions, whereas Zn accumulation and translocation was found in both ultramafic and non-ultramafic accessions.

Conclusions

Ni accumulation and translocation was restricted to ultramafic populations of both species, whereas it is a species-wide trait for Zn. This study provides new and comprehensive information on the natural variation of hyperaccumulation of Ni, Zn and Cd in N. kovatsii and N. praecox.

     

Palatability of Thlaspi caerulescens for snails: influence of zinc and glucosinolates

* The hypothesis that zinc (Zn) hyperaccumulation defends Thlaspi caerulescens against herbivores is tested with the snail Helix aspersa. We investigated the effects of leaf zinc, cadmium, glucosinolate, nitrogen and dry matter concentrations on the feeding preferences of snails. * Four T. caerulescens populations from southern France (two from metalliferous and two from normal soils) were grown on low- and high-Zn soils to obtain contrasting leaf Zn concentrations. Plants were also collected in the field, and binary feeding choices involving low- and high-Zn leaves were conducted. * Foliar Zn, Cd, N and dry matter concentrations did not affect the feeding choices of snails, whereas glucosinolate had a significant negative effect on herbivore preferences. Compared with metallicolous plants, nonmetallicolous ones appeared to be better protected against snails, whatever their Zn concentration. * These results do not support the defence hypothesis, as glucosinolates appear to decrease the degree of herbivory when Zn does not.