Damien Blaudez

Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity.

BackgroundThe Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters.ResultsRepresentative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed.ConclusionIn the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members.

CNGCs: prime targets of plant cyclic nucleotide signalling?

Cyclic nucleotide-gated channels (CNGCs) are a recently identified family of plant ion channels. They show a high degree of similarity to Shaker-type voltage-gated channels and contain a C-terminal cyclic nucleotide-binding domain with an overlapping calmodulin-binding domain. Heterologously expressed plant CNGCs show activation by cyclic nucleotides and permeability to monovalent and divalent cations. In plants, downstream effectors of cyclic nucleotide signals have so far remained obscure, and CNGCs might be their prime targets. The unique position of CNGCs as ligand-gated Ca(2+)-permeable channels suggests that they function at key sites where cyclic nucleotide and Ca(2+) signalling pathways interact. Such processes include plant defence responses, and two recently characterized Arabidopsis mutants in CNGC genes indeed show altered pathogen responses.

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd2+-uptake kinetics were characterized by a smooth, non-saturating curve that could be dissected into linear and saturable components. The linear-uptake kinetic component was interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component was the result of carrier-mediated transport across the plasma membrane. Cell-wall-bound Cd was almost completely removed during desorption from cell-wall preparations. Cd2+ desorption from intact mycelium was found to be a function of time involving three compartments corresponding in series to cell wall (50%), cytoplasm (30%) and vacuole (20%), when mycelia were exposed to a 0.05 microM Cd concentration. At 4 degrees C, most of the Cd recovered was due to the cell-wall-bound fraction, suggesting that transport across the plasma membrane is a metabolically mediated process. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Cd accumulation in P. involutus mycelia by up to 28%, which indicates that transport of Cd2+ was partially dependent on the membrane potential. Cd2+ uptake into symplasm is linked to Ca2+ transport, as revealed by the inhibition of Cd accumulation by the Ca2+ ionophore A23187. The present work demonstrates the ability of the ectomycorrhizal fungus P. involutus to take up and further accumulate Cd in different compartments. Binding of Cd onto cell walls and accumulation of Cd in the vacuolar compartment may be regarded as two essential metal-detoxification mechanisms. These data represent a first step towards the understanding of metal-tolerance mechanisms in mycorrhizal fungi.

Poplar Metal Tolerance Protein 1 Confers Zinc Tolerance and Is an Oligomeric Vacuolar Zinc Transporter with an Essential Leucine Zipper Motif

Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. Here, we describe the identification, characterization, and localization of PtdMTP1, a member of the CDF family from the hybrid poplar Populus trichocarpa × Populus deltoides. PtdMTP1 is expressed constitutively and ubiquitously, although at low levels. Heterologous expression in yeast showed that PtdMTP1 was able to complement the hypersensitivity of mutant strains to Zn but not to other metals, including Cd, Co, Mn, and Ni. PtdMTP1 fused to green fluorescent protein localized to the vacuolar membrane both in yeast and in plant cells, consistent with a function of PtdMTP1 in zinc sequestration. Overexpression of PtdMTP1 in Arabidopsis confers Zn tolerance. We show that PtdMTP1, when expressed in yeast and Arabidopsis, forms homooligomers, a novel feature of CDF members. Oligomer formation is disrupted by reducing agents, indicating possible disulfide bridge formation. PtdMTP1 also contains a conserved Leu zipper motif. Although not necessary for oligomer formation, Leu residues within this motif are required for PtdMTP1 functional activity.

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganese-hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11–EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.

Differential responses of ectomycorrhizal fungi to heavy metals in vitro.

Thirty-nine ectomycorrhizal isolates of Paxillus involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and S. variegatus were tested on cadmium, copper, nickel and zinc amended media to determine their in vitro tolerance, measured as inhibition of biomass production. Twenty-one isolates were from heavy metal polluted sites, whereas the others were from non-contaminated soils. There was a strong interspecific variation in metal tolerance. S. luteus, S. variegatus and P. tinctorius were more tolerant of Cu, Cd and Zn when compared with P. involutus, whereas the reverse was true for Ni. A high intraspecific heterogeneity in metal tolerance was also found. EC 50 values for isolates originating from polluted sites were not statistically different from EC 50 values for isolates originating from non-contaminated sites. The findings are discussed in relation to the potential benefits of ectomycorrhizal fungi in protecting their host plants from metal contamination.

An update on nutrient transport processes in ectomycorrhizas

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.

Structure and expression profile of the phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa

Gene networks involved in inorganic phosphate (Pi) acquisition and homeostasis in woody perennial species able to form mycorrhizal symbioses are poorly known. Here, we describe the features of the 12 genes coding for Pi transporters of the Pht1 family in poplar (Populus trichocarpa). Individual Pht1 transporters play distinct roles in acquiring and translocating Pi in different tissues of mycorrhizal and nonmycorrhizal poplar during different growth conditions and developmental stages. Pi starvation triggered the up-regulation of most members of the Pht1 family, especially PtPT9 and PtPT11. PtPT9 and PtPT12 showed a striking up-regulation in ectomycorrhizas and endomycorrhizas, whereas PtPT1 and PtPT11 were strongly down-regulated. PtPT10 transcripts were highly abundant in arbuscular mycorrhiza (AM) roots only. PtPT8 and PtPT10 are phylogenetically associated to the AM-inducible Pht1 subfamily I. The analysis of promoter sequences revealed conserved motifs similar to other AM-inducible orthologs in PtPT10 only. To gain more insight into gene regulatory mechanisms governing the AM symbiosis in woody plant species, the activation of the poplar PtPT10 promoter was investigated and detected in AM of potato (Solanum tuberosum) roots. These results indicated that the regulation of AM-inducible Pi transporter genes is conserved between perennial woody and herbaceous plant species. Moreover, poplar has developed an alternative Pi uptake pathway distinct from AM plants, allowing ectomycorrhizal poplar to recruit PtPT9 and PtPT12 to cope with limiting Pi concentrations in forest soils.

Genome-wide analysis of plant metal transporters, with an emphasis on poplar.

The specific transport of metal ions, mediated by membrane-localized metal transporters, is of fundamental importance in all eukaryotes. Genome-wide analysis of metal transporters was undertaken, making use of whole genome sequences of the green alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the monocots rice and sorghum, and the dicots Arabidopsis thaliana, poplar, grapevine, as well as of the yeast Saccharomyces cerevisiae. A repertoire of 430 metal transporters was found in total across eight photosynthetic plants, as well as in S. cerevisiae. Seventy-two full-length metal transporter genes were identified in the Populus genome alone, which is the largest number of metal transporters genes identified in any single species to date. Diversification of some transporter family gene clusters appears to have occurred in a lineage-specific manner. Expression analysis of Populus metal transporters indicates that some family members show tissue-specific transcript abundance. Taken together, the data provide a picture into the diversification of these important gene families.

Taxonomic and functional diversity of Streptomyces in a forest soil

In this work we report the isolation and the characterization of 79 Streptomyces isolates from a French forest soil. The 16S rRNA gene phylogeny indicated that a great diversity of Streptomyces was present in this soil, with at least nine different and potentially new species. Growth plate assays showed that most Streptomyces lineages exhibit cellulolytic and hemicellulolytic capacities and potentially participate in wood decomposition. Molecular screening for a specific hydrogenase also indicated a widespread potential for atmospheric H2 uptake. Co-culture experiments with representative strains showed antagonistic effects between Streptomyces of the same population and between Streptomyces and various fungi. Interestingly, in certain conditions, growth promotion of some fungi also occurred. We conclude that in forest soil, Streptomyces populations exhibit many important functions involved in different biogeochemical cycles and also influence the structure of soil microbial communities.