RESUMEN
As a drastic environmental change, metal pollution may promote the rapid evolution of genetic adaptations contributing to metal tolerance. In Arabidopsis halleri, genetic bases of zinc (Zn) and cadmium (Cd) tolerance have been uncovered only in a metallicolous accession, although tolerance is species-wide. The genetic determinants of Zn and Cd tolerance in a nonmetallicolous accession were thus investigated for the first time. The genetic architecture of tolerance was investigated in a nonmetallicolous population (SK2) by using first backcross progeny obtained from crosses between SK2 and Arabidopsis lyrata petraea, a nonmetallophyte species. Only one significant and common quantitative trait locus (QTL) region was identified explaining 22.6% and 31.2% of the phenotypic variation for Zn and Cd tolerance, respectively. This QTL co-localized with HEAVY METAL ATPASE 4 (AhHMA4), which was previously validated as a determinant of Zn and Cd tolerance in a metallicolous accession. Triplication and high expression of HMA4 were confirmed in SK2. In contrast, gene duplication and high expression of METAL TOLERANT PROTEIN 1A (MTP1A), which was previously associated with Zn tolerance in a metallicolous accession, were not observed in SK2. Overall, the results support the role of HMA4 in tolerance capacities of A. halleri that may have pre-existed in nonmetallicolous populations before colonization of metal-polluted habitats. Preadaptation to metal-contaminated sites is thus discussed.
Asunto(s)
Adaptación Fisiológica/genética , Arabidopsis/genética , Cadmio/toxicidad , Ecotipo , Contaminación Ambiental , Actividades Humanas , Sitios de Carácter Cuantitativo/genética , Zinc/toxicidad , Adaptación Fisiológica/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Cruzamientos Genéticos , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Humanos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Cadmio/toxicidad , Segregación Cromosómica , Estrés Oxidativo/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Calcio/farmacología , Clonación Molecular , Simulación por Computador , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Estudios de Asociación Genética , Marcadores Genéticos , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Sitios de Carácter CuantitativoRESUMEN
Arabidopsis halleri is a model plant for Zn and Cd hyperaccumulation. The objective of this study was to determine the relationship between the chemical forms of Cd, its distribution in leaves, and Cd accumulation and tolerance. An interspecific cross was carried out between A. halleri and the non-tolerant and non-hyperaccumulating relative A. lyrata providing progenies segregating for Cd tolerance and accumulation. Cd speciation and distribution were investigated using X-ray absorption spectroscopy and microfocused X-ray fluorescence. In A. lyrata and non-tolerant progenies, Cd was coordinated by S atoms only or with a small contribution of O groups. Interestingly, the proportion of O ligands increased in A. halleri and tolerant progenies, and they were predominant in most of them, while S ligands were still present. Therefore, the binding of Cd with O ligands was associated with Cd tolerance. In A. halleri, Cd was mainly located in the xylem, phloem, and mesophyll tissue, suggesting a reallocation process for Cd within the plant. The distribution of the metal at the cell level was further discussed. In A. lyrata, the vascular bundles were also Cd enriched, but the epidermis was richer in Cd as compared with the mesophyll. Cd was identified in trichomes of both species. This work demonstrated that both Cd speciation and localization were related to the tolerance character of the plant.
Asunto(s)
Arabidopsis/metabolismo , Cadmio/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Cadmio/toxicidad , Hibridación Genética , Fenotipo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Especificidad de la Especie , Espectrometría por Rayos X , Sincrotrones , Distribución Tisular , Xilema/efectos de los fármacos , Xilema/genética , Xilema/metabolismo , Zinc/metabolismoRESUMEN
Certain molecular mechanisms of Cd tolerance and accumulation have been identified in the model species Arabidopsis halleri, while intraspecific variability of these traits and the mechanisms of shoot detoxification were little addressed. The Cd tolerance and accumulation of metallicolous and non-metallicolous A. halleri populations from different genetic units were tested in controlled conditions. In addition, changes in shoot cell wall composition were investigated using Fourier transform infrared spectroscopy. Indeed, recent works on A. halleri suggest Cd sequestration both inside cells and in the cell wall/apoplast. All A. halleri populations tested were hypertolerant to Cd, and the metallicolous populations were on average the most tolerant. Accumulation was highly variable between and within populations, and populations that were non-accumulators of Cd were identified. The effect of Cd on the cell wall composition was quite similar in the sensitive species A. lyrata and in A. halleri individuals; the pectin/polysaccharide content of cell walls seems to increase after Cd treatment. Nevertheless, the changes induced by Cd were more pronounced in the less tolerant individuals, leading to a correlation between the level of tolerance and the extent of modifications. This work demonstrated that Cd tolerance and accumulation are highly variable traits in A. halleri, suggesting adaptation at the local scale and involvement of various molecular mechanisms. While in non-metallicolous populations drastic modifications of the cell wall occur due to higher Cd toxicity and/or Cd immobilization in this compartment, the increased tolerance of metallicolous populations probably involves other mechanisms such as vacuolar sequestration.
Asunto(s)
Arabidopsis/genética , Cadmio/metabolismo , Pared Celular/efectos de los fármacos , Variación Genética , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Biomasa , Cadmio/toxicidad , Pared Celular/metabolismo , Hibridación Genética , Hidroponía , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Análisis de Componente Principal , Especificidad de la Especie , Espectroscopía Infrarroja por Transformada de Fourier , Estrés FisiológicoRESUMEN
On sols highly polluted by trace metallic elements the majority of plant species are excluders, limiting the entry and the root to shoot translocation of trace metals. However a rare class of plants called hyperaccumulators possess remarkable adaptation because those plants combine extremely high tolerance degrees and foliar accumulation of trace elements. Hyperaccumulators have recently gained considerable interest, because of their potential use in phytoremediation, phytomining and biofortification. On a more fundamental point of view hyperaccumulators of trace metals are case studies to understand metal homeostasis and detoxification mechanisms. Hyperaccumulation of trace metals usually depends on the enhancement of at least four processes, which are the absorption from the soil, the loading in the xylem in the roots and the unloading from the xylem in the leaves and the detoxification in the shoot. Cadmium is one of the most toxic trace metallic elements for living organisms and its accumulation in the environment is recognized as a worldwide concern. To date, only nine species have been recognized as Cd hyperaccumulators that is to say able to tolerate and accumulate more than 0.01 % Cd in shoot dry biomass. Among these species, four belong to the Brassicaceae family with Arabidopsis halleri and Noccaea caerulescens being considered as models. An update of our knowledge on the evolution of hyperaccumulators will be presented here.
Asunto(s)
Cadmio/metabolismo , Cadmio/toxicidad , Plantas/efectos de los fármacos , Plantas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismoRESUMEN
Genome scans based on anonymous Amplified Fragment Length Polymorphism (AFLP) markers scattered throughout the genome are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation in natural populations. A shortcoming of this approach is that despite its efficiency to detect signatures of selection, it can hardly help pinpoint the specific genomic region(s), gene(s), or mutation(s) targeted by selection. Here, we present two methods to be undertaken after performing an AFLP-based genome scan to easily obtain the sequences of AFLP loci detected as outliers by population genomics approaches. The first one is based on the gel excision of the target AFLP fragment, after simplification of the AFLP fingerprint and separation of the fragments by migration. The second one is a combination of classical AFLP protocol and 454 pyrosequencing.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Secuencia de Bases/genética , Genoma , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Adaptación Fisiológica/genética , Animales , Bacterias , Electroforesis en Gel de Poliacrilamida , Hongos , Sitios Genéticos , Especiación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Plantas , Polimorfismo Genético , Selección GenéticaRESUMEN
There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Félix-de-Pallières, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Félix-de-Pallières) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.
Asunto(s)
Adenosina Trifosfatasas/genética , Cadmio/metabolismo , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Thlaspi/enzimología , Adenosina Trifosfatasas/metabolismo , Proteínas de Plantas/metabolismo , Thlaspi/genética , Thlaspi/metabolismoRESUMEN
The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²âº ions.
Asunto(s)
Aminoaciltransferasas/metabolismo , Arabidopsis/enzimología , Fitoquelatinas/biosíntesis , Thlaspi/enzimología , Secuencia de Aminoácidos , Aminoaciltransferasas/química , Aminoaciltransferasas/aislamiento & purificación , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Cadmio/metabolismo , Quelantes/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Datos de Secuencia Molecular , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Thlaspi/genética , Thlaspi/metabolismo , Zinc/metabolismoRESUMEN
We estimated the level of quantitative polymorphism for zinc (Zn) tolerance in neighboring metallicolous and nonmetallicolous populations of Arabidopsis halleri and tested the hypothesis that divergent selection has shaped this polymorphism. A short-term hydroponic test was used to capture the quantitative polymorphism present between edaphic types, among and within populations. We measured six morphological and physiological traits on shoots and roots to estimate the response of A. halleri to Zn. In order to assess the adaptive value of Zn tolerance polymorphism, we compared differentiation of quantitative traits with that of molecular markers. Zinc tolerance of metallicolous populations was, on average, higher than that of nonmetallicolous populations according to the morphological and physiological traits measured. Phenotypic variability within edaphic types was very high and mainly explained by polymorphism among individuals within populations. Genetic differentiation for photosystem II yield of leaves (a measure of photosynthetic efficiency) was greater than the differentiation for microsatellite and thus, probably shaped by divergent selection. Overall, these results suggest that, in the sampled populations, Zn tolerance has been increased in metallicolous populations through selection on standing genetic variation within local nonmetallicolous ancestral populations.
Asunto(s)
Adaptación Biológica , Arabidopsis/genética , Fotosíntesis/genética , Polimorfismo Genético , Selección Genética , Zinc , Arabidopsis/metabolismo , Repeticiones de Microsatélite , Fenotipo , Complejo de Proteína del Fotosistema II/genética , Raíces de Plantas , Brotes de la PlantaRESUMEN
Pollution by heavy metals is one of the strongest environmental constraints in human-altered environments that only a handful of species can cope with. Identifying the genes conferring to those species the ability to grow in polluted areas is a first step towards a global understanding of the evolutionary processes involved and will eventually improve phytoremediation practices. We used a genome-scan approach to detect loci under divergent selection among four populations of Arabidopsis halleri growing on either polluted or nonpolluted habitats. Based on a high density of amplified fragment length polymorphism (AFLP) markers (820 AFLP markers, i.e. approximately 1 marker per 0.3Mb), evidence for selection was found for some markers in every sampled population. Four loci departed from neutrality in both metallicolous populations and thus constitute high-quality candidates for general adaptation to pollution. Interestingly, some candidates differed between the two metallicolous populations, suggesting the possibility that different loci may be involved in adaptation in the different metallicolous populations.