The Five AhMTP1 Zinc Transporters Undergo Different Evolutionary Fates towards Adaptive Evolution to Zinc Tolerance in Arabidopsis halleri

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn2+/H+ antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiæ zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance

Does Speciation between Arabidopsis halleri and Arabidopsis lyrata Coincide with Major Changes in a Molecular Target of Adaptation?

Ever since Darwin proposed natural selection as the driving force for the origin of species, the role of adaptive processes in speciation has remained controversial. In particular, a largely unsolved issue is whether key divergent ecological adaptations are associated with speciation events or evolve secondarily within sister species after the split. The plant Arabidopsis halleri is one of the few species able to colonize soils highly enriched in zinc and cadmium. Recent advances in the molecular genetics of adaptation show that the physiology of this derived ecological trait involves copy number expansions of the AhHMA4 gene, for which orthologs are found in single copy in the closely related A. lyrata and the outgroup A. thaliana. To gain insight into the speciation process, we ask whether adaptive molecular changes at this candidate gene were contemporary with important stages of the speciation process. We first inferred the scenario and timescale of speciation by comparing patterns of variation across the genomic backgrounds of A. halleri and A. lyrata. Then, we estimated the timing of the first duplication of AhHMA4 in A. halleri. Our analysis suggests that the historical split between the two species closely coincides with major changes in this molecular target of adaptation in the A. halleri lineage. These results clearly indicate that these changes evolved in A. halleri well before industrial activities fostered the spread of Zn- and Cd-polluted areas, and suggest that adaptive processes related to heavy-metal homeostasis played a major role in the speciation process

Intraspecific variation of metal preference patterns for hyperaccumulation in Thlaspi caerulescens: evidence from binary metal exposures.

Metal preferences with regard to accumulation were compared between populations of the heavy metal hyperaccumulator Thlaspi caerulescens, originating from calamine, serpentine and non-metalliferous soils. Plants were exposed for 3 weeks to factorial combinations of concentrations of different metals in binary mixture in hydroponics. The nature and degree of the interactions varied significantly between populations. In the calamine, non-Cd/Ni-hyperaccumulating population, La Calamine, there were no one-sided or mutual antagonistic interactions among the metals with regard to their accumulation in the plant. In three other populations capable of Cd and Ni hyperaccumulation, from calamine, serpentine and non-metalliferous soil respectively, there were one-sided or mutual antagonistic interactions between Cd and Zn, Cd and Ni, and Ni and Zn, possibly resulting from competition for transporters involved in uptake or plant-internal transport. Significant synergistic interactions, probably resulting from regulation of transporter expression, were also found, particularly in the La Calamine population. All the populations seemed to express a more or less Zn-specific high-affinity system. The serpentine and the non-metallicolous populations seemed to posses low-affinity systems with a preference for Cd and Zn over Ni, one of which may be responsible for the Ni hyperaccumulation of the serpentine population in its natural environment. The calamine population from Ganges also seemed to express a strongly Cd-specific high-affinity system which is in part responsible for the Cd-hyperaccumulation phenotype exhibited by this population in its natural environment. © 2007 The Author(s)

C. PRESL) at the transcriptional level.

This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil

Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4

Little is known about the types of mutations underlying the evolution of species-specific traits. The metal hyperaccumulator Arabidopsis halleri has the rare ability to colonize heavy-metal-polluted soils, and, as an extremophile sister species of Arabidopsis thaliana, it is a powerful model for research on adaptation. A. halleri naturally accumulates and tolerates leaf concentrations as high as 2.2% zinc and 0.28% cadmium in dry biomass. On the basis of transcriptomics studies, metal hyperaccumulation in A. halleri has been associated with more than 30 candidate genes that are expressed at higher levels in A. halleri than in A. thaliana. Some of these genes have been genetically mapped to broad chromosomal segments of between 4 and 24 cM co-segregating with Zn and Cd hypertolerance. However, the in planta loss-of-function approaches required to demonstrate the contribution of a given candidate gene to metal hyperaccumulation or hypertolerance have not been pursued to date. Using RNA interference to downregulate HMA4 (HEAVY METAL ATPASE 4) expression, we show here that Zn hyperaccumulation and full hypertolerance to Cd and Zn in A. halleri depend on the metal pump HMA4. Contrary to a postulated global trans regulatory factor governing high expression of numerous metal hyperaccumulation genes, we demonstrate that enhanced expression of HMA4 in A. halleri is attributable to a combination of modified cis-regulatory sequences and copy number expansion, in comparison to A. thaliana. Transfer of an A. halleri HMA4 gene to A. thaliana recapitulates Zn partitioning into xylem vessels and the constitutive transcriptional upregulation of Zn deficiency response genes characteristic of Zn hyperaccumulators. Our results demonstrate the importance of cis-regulatory mutations and gene copy number expansion in the evolution of a complex naturally selected extreme trait. The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils and for bio-fortification

Hard Selective Sweep and Ectopic Gene Conversion in a Gene Cluster Affording Environmental Adaptation

13 Págs., 7 Figs., 6 Tabls. 9 Pag., 8 Fig. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Among the rare colonizers of heavy-metal rich toxic soils, Arabidopsis halleri is a compelling model extremophile, physiologically distinct from its sister species A. lyrata, and A. thaliana. Naturally selected metal hypertolerance and extraordinarily high leaf metal accumulation in A. halleri both require Heavy Metal ATPase4 (HMA4) encoding a PIB-type ATPase that pumps Zn2+ and Cd2+ out of specific cell types. Strongly enhanced HMA4 expression results from a combination of gene copy number expansion and cis-regulatory modifications, when compared to A. thaliana. These findings were based on a single accession of A. halleri. Few studies have addressed nucleotide sequence polymorphism at loci known to govern adaptations. We thus sequenced 13 DNA segments across the HMA4 genomic region of multiple A. halleri individuals from diverse habitats. Compared to control loci flanking the three tandem HMA4 gene copies, a gradual depletion of nucleotide sequence diversity and an excess of low-frequency polymorphisms are hallmarks of positive selection in HMA4 promoter regions, culminating at HMA4-3. The accompanying hard selective sweep is segmentally eclipsed as a consequence of recurrent ectopic gene conversion among HMA4 protein-coding sequences, resulting in their concerted evolution. Thus, HMA4 coding sequences exhibit a network-like genealogy and locally enhanced nucleotide sequence diversity within each copy, accompanied by lowered sequence divergence between paralogs in any given individual. Quantitative PCR corroborated that, across A. halleri, three genomic HMA4 copies generate overall 20- to 130-fold higher transcript levels than in A. thaliana. Together, our observations constitute an unexpectedly complex profile of polymorphism resulting from natural selection for increased gene product dosage. We propose that these findings are paradigmatic of a category of multi-copy genes from a broad range of organisms. Our results emphasize that enhanced gene product dosage, in addition to neo- and sub-functionalization, can account for the genomic maintenance of gene duplicates underlying environmental adaptation. © 2013 Hanikenne et al.Funding was provided by, the Heisenberg Fellowship Kr1967/4-1, InP "PHIME" FOOD-CT-2006-016253, German Research Foundation Kr1967/3-2 and SPP1529 "ADAPTOMICS" Kr1967/10-1 (UK), European Union RTN "METALHOME" HPRN-CT-2002-00243 (SC, UK), the Max Planck Institute for Chemical Ecology, Jena, Germany (JK), Fonds de la Recherche Scientifique FNRS 2.4540.06, 2.4583.08 and 2.4581.10, "Fonds Spéciaux du Conseil de la Recherche", University of Liège (PM, MH). MH was a Research Associate of the FNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Intrinsically Photosensitive Retinal Ganglion Cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light in the absence of all rod and cone photoreceptor input. The existence of these ganglion cell photoreceptors, although predicted from observations scattered over many decades, was not established until it was shown that a novel photopigment, melanopsin, was expressed in retinal ganglion cells of rodents and primates. Phototransduction in mammalian ipRGCs more closely resembles that of invertebrate than vertebrate photoreceptors and appears to be mediated by transient receptor potential channels. In the retina, ipRGCs provide excitatory drive to dopaminergic amacrine cells and ipRGCs are coupled to GABAergic amacrine cells via gap junctions. Several subtypes of ipRGC have been identified in rodents based on their morphology, physiology and expression of molecular markers. ipRGCs convey irradiance information centrally via the optic nerve to influence several functions including photoentrainment of the biological clock located in the hypothalamus, the pupillary light reflex, sleep and perhaps some aspects of vision. In addition, ipRGCs may also contribute irradiance signals that interface directly with the autonomic nervous system to regulate rhythmic gene activity in major organs of the body. Here we review the early work that provided the motivation for searching for a new mammalian photoreceptor, the ground-breaking discoveries, current progress that continues to reveal the unusual properties of these neuron photoreceptors, and directions for future investigation