Comparative landscape genetic analyses show a Belgian motorway to be a gene flow barrier for red deer (Cervus elaphus), but not wild boars (Sus scrofa).

While motorways are often assumed to influence the movement behaviour of large mammals, there are surprisingly few studies that show an influence of these linear structures on the genetic make-up of wild ungulate populations. Here, we analyse the spatial genetic structure of red deer (Cervus elaphus) and wild boars (Sus scrofa) along a stretch of motorway in the Walloon part of Belgium. Altogether, 876 red deer were genotyped at 13 microsatellite loci, and 325 wild boars at 14 loci. In the case of the red deer, different genetic clustering tools identified two genetic subpopulations whose borders matched the motorway well. Conversely, no genetic structure was identified in the case of the wild boar. Analysis of isolation-by-distance patterns of pairs of individuals on the same side and on different sides of the motorway also suggested that the road was a barrier to red deer, but not to wild boar movement. While telemetry studies seem to confirm that red deer are more affected by motorways than wild boar, the red deer sample size was also much larger than that of the wild boars. We therefore repeated the analysis of genetic structure in the red deer with randomly sub-sampled data sets of decreasing size. The power to detect the genetic structure using clustering methods decreased with decreasing sample size.

Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice.

An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals.

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms.

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agre's team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.

Genetic structure and assignment tests demonstrate illegal translocation of red deer (Cervus elaphus) into a continuous population.

Molecular forensic methods are being increasingly used to help enforce wildlife conservation laws. Using multilocus genotyping, illegal translocation of an animal can be demonstrated by excluding all potential source populations as an individual's population of origin. Here, we illustrate how this approach can be applied to a large continuous population by defining the population genetic structure and excluding suspect animals from each identified cluster. We aimed to test the hypothesis that recreational hunters had illegally introduced a group of red deer into a hunting area in Luxembourg. Reference samples were collected over a large area in order to test the possibility that the suspect individuals might be recent immigrants. Due to isolation-by-distance relationships in the data set, inferring the number of genetic clusters using Bayesian methods was not straightforward. Biologically meaningful clusters were only obtained by simultaneously analysing spatial and genetic information using the program baps 4.1. We inferred the presence of three genetic clusters in the study region. Using partial Mantel tests, we detected barriers to gene flow other than distance, probably created by a combination of urban areas, motorways and a river valley used for viticulture. The four focal animals could be excluded with a high certainty from the three genetic subpopulations and it was therefore likely that they had been released illegally.

Aquaporins constitute a large and highly divergent protein family in maize.

Aquaporins (AQPs) are an ancient family of channel proteins that transport water and neutral solutes through a pore and are found in all eukaryotes and most prokaryotes. A comparison of the amino acid sequences and phylogenetic analysis of 31 full-length cDNAs of maize (Zea mays) AQPs shows that they comprise four different groups of highly divergent proteins. We have classified them as plasma membrane intinsic proteins (PIPs), tonoplast intrinsic proteins, Nod26-like intrinsic proteins, and small and basic intrinsic proteins. Amino acid sequence identities vary from 16% to 100%, but all sequences share structural motifs and conserved amino acids necessary to stabilize the two loops that form the aqueous pore. Most divergent are the small and basic integral proteins in which the first of the two highly conserved Asn-Pro-Ala motifs of the pore is not conserved, but is represented by alanine-proline-threonine or alanine-proline-serine. We present a model of ZmPIP1-2 based on the three-dimensional structure of mammalian AQP1. Tabulation of the number of times that the AQP sequences are found in a collection of databases that comprises about 470,000 maize cDNAs indicates that a few of the maize AQPs are very highly expressed and many are not abundantly expressed. The phylogenetic analysis supports the interpretation that the divergence of PIPs through gene duplication occurred more recently than the divergence of the members of the other three subfamilies. This study opens the way to analyze the function of the proteins in Xenopus laevis oocytes, determine the tissue specific expression of the genes, recover insertion mutants, and determine the in planta function.

Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity.

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins.

Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells.

Changes in vacuolar structure and the expression at the RNA level of a tonoplast aquaporin (BobTIP26-1) were examined in cauliflower (Brassicaoleracea L. var. botrytis) under water-stress conditions. Gradual drying out of slices of cauliflower floret tissue caused its collapse, with a shrinkage in tissue and cell volumes and an apparent vesiculation of the central vacuole, whereas osmotic stress resulted in plasmolysis with a collapse of the cytoplasm and the central vacuole within. Osmotic stress caused a rapid and substantial increase in BobTIP26 mRNA in slices of floret tissue. Exposure of tissue slices to a regime of desiccation showed a slower but equally large rise in BobTIP26 mRNA followed by a rapid decline upon rehydration. In situ hybridization showed that BobTIP26-2 mRNA is expressed most highly in meristematic and expanding cells of the cauliflower florets and that desiccation strongly increased the expression in those cells and in differentiated cells near the xylem vessels. These data indicate that under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells.

Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation.

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin gamma-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean alpha-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

High expression of the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.

Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein.

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

Expression of an Arabidopsis plasma membrane aquaporin in Dictyostelium results in hypoosmotic sensitivity and developmental abnormalities.

The rd28 gene of Arabidopsis thaliana encodes a water channel protein, or aquaporin, of the plasma membrane. A construct in which transcription of the rd28 cDNA is controlled by the Dictyostelium actin15 promoter was transformed into Dictyostelium discoideum cells. Transformants contained RD28 protein in their plasma membranes. When shifted to a low-osmotic-strength buffer, cells expressing rd28 swelled rapidly and burst, indicating that the plant aquaporin allowed rapid water entry in the amoebae. The rate of osmotic lysis was a function of the osmotic pressure of the buffer. We also selected transformants in which the expression of the rd28 cDNA is driven by the promoter of the prespore cotB gene. These transformants accumulated rd28 mRNA uniquely in prespore cells. In low-osmotic-strength buffer, the cotB::rd28 cells aggregated and formed normally proportioned slugs but failed to form normal fruiting bodies. The number of spores was reduced 20-fold, and the stalks of the fruiting bodies were abnormally short. The consequences of expressing RD28 in prespore cells could be partially overcome by increasing the osmolarity of the medium. Under these conditions, the cotB::rd28 cells formed fruiting bodies of more normal appearance, and the number of viable spores increased slightly. Because prespore cells have to shrink and dehydrate to form spores, it was not unexpected that expression of an aquaporin would disrupt this process, but it was surprising to find that stalk differentiation was also affected by expression of rd28 in prespore cells. It appears that osmotic stress on prespore cells alters their ability to signal terminal differentiation in prestalk cells. The results provide independent confirmation that plant aquaporins can function in the cells of other organisms, and that D. discoideum can be used to study the properties of these water channels.

Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site.

The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha-TIP showed that the TIPs are differentially expressed.

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles.

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F1-ATPase beta-subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial beta-presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immunodetection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.

Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration.

The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility. We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species. The coding sequence of T-urf13 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Plants expressing high levels of URF13 were methomyl sensitive. Subcellular analysis indicated that URF13 is mainly associated with the mitochondria. Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urf13c construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction. Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria. No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria. There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.

Aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from Euphorbia characias.

Metabolic studies on Phytomonas sp. isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias indicate that glucose is the preferred energy and carbon substrate during logarithmic growth. In stationary phase cells glucose consumption was dramatically reduced. Glucose consumption and end-product formation were measured on logarithmically growing cells, both under aerobic (air and 95% O2/5% CO2) and anaerobic (95% N2/5% CO2 and 100% N2) conditions. The rate of glucose consumption slightly increased under anaerobic conditions indicating that Phytomonas lacks a 'reverse Pasteur' effect contrary to the situation encountered in Leishmania major. Major end-products of glucose catabolism under aerobic conditions, detected by enzymatic and NMR measurements, were acetate, ethanol and carbon dioxide and under anaerobic conditions ethanol, glycerol and carbon dioxide. Smaller amounts of pyruvate, succinate, L-malate, L-lactate, phosphoenolpyruvate, alanine and aspartate were also detected.

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria.

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Protein transport into mitochondria is conserved between plant and yeast species.

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.