OmGOGAT-disruption in the ericoid mycorrhizal fungus Oidiodendron maius induces reorganization of the N pathway and reduces tolerance to heavy-metals.

Mycorrhizal fungi are key mediators of soil-to-plant movement of mineral nutrients, including essential and non-essential metals. In soil conditions that facilitate mobilization of metal ions, potentially toxic metals can interfere with nitrogen metabolism in both plants and microorganisms. Less is known about possible relationships between nitrogen metabolism and responses to heavy metals. Aim of this study was to investigate this aspect in the ericoid mycorrhizal fungus Oidiodendron maius strain Zn, a metal tolerant ascomycete. Growth of O. maius Zn on zinc and cadmium containing media was significantly affected by the nitrogen source. Screening of a library of O. maius Zn random genetic transformants for sensitivity to heavy metals (zinc and cadmium) and oxidative stress (menadione) yielded a mutant strain that carried a partial deletion of the glutamate synthase (NADH-GOGAT EC 1.4.1.14) gene and its adjacent gene, the APC15 subunit of the anaphase promoting complex. Comparison of WT and OmGOGAT-OmAPC15 mutant strains indicated an impaired N-metabolism and altered stress tolerance, and assays on the OmAPC15-recomplemented strains ascribed the observed phenotypes to the deletion in the OmGOGAT gene. OmGOGAT disruption modified the nitrogen pathway, with a strong reduction of the associated glutamine synthetase (GS, EC 6.3.1.2) activity and an up-regulation of the alternative NADP-glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) pathway for glutamate biosynthesis. Unless they were supplemented with glutamine, O. maius Zn transformants lacking OmGOGAT were very sensitive to zinc. These results highlight the importance of nitrogen metabolism not only for nitrogen assimilation and transformation, but also for stress tolerance. For mycorrhizal fungi, such as O. maius, this may bear consequences not only to the fungus, but also to the host plant.

The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis.

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Increasing detectivity of polarization modulation infrared reflection-absorption spectroscopy for the study of ultrathin films deposited on various substrates.

In this paper, we present a simple way to increase the sensitivity of polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) for the study of ultrathin films deposited on dielectric and semiconductor substrates. The enhancement of the absorption band intensity is obtained by reducing the signal arising from the substrate. This is achieved by adding a polarizer after the sample in order to balance the polarized reflectivities of the sample. As a consequence, the contribution of the film to the PM-IRRAS signal is increased relative to that of the substrate. An enhancement factor of about 10 has been obtained for ultrathin organic films deposited on glass and spread at the air-water interface. This method has also allowed the study of the very thin native oxide layer present on silicon without the need for the reference spectrum of bare silicon.

A phospholipid bilayer supported under a polymerized Langmuir film.

Phospholipid single bilayers supported on a hydrophilic solid substrate are extensively used in the study of the interaction between model membranes and proteins or polypeptides. In this article, the formation of a single dimyristoylphosphatidylcholine (DMPC) bilayer under an octadecyltrimethoxysilane (OTMS) polymerized Langmuir monolayer at the air-water interface is followed by Brewster angle microscopy (BAM) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The formation of the bilayer is initiated by injection of dimyristoylphosphatidylcholine small unilamellar vesicles into the aqueous subphase. Brewster angle microscopy allows visualization of the kinetics of formation and the homogeneity of the bilayer. Spectral simulations of the polarization-modulated infrared reflection absorption spectroscopy spectra reveal that the bilayer thickness is 39 +/- 5 A. This system constitutes the first example of a phospholipid bilayer on a "nanoscopic" support and opens the way to studies involving supported bilayers using powerful experimental techniques such as x-ray reflectivity, vibrational spectroscopies, or Brewster angle microscopy.

Structure of a fusion peptide analogue at the air-water interface, determined from surface activity, infrared spectroscopy and scanning force microscopy.

We have investigated a point mutant of the HIV-1 fusion peptide in a compressed monolayer at the air-water interface. A variety of surface sensitive techniques were applied to study structural features under conditions mimicking the hydrophobic/hydrophilic environment of a biomembrane. Possible partitioning into the aqueous bulk phase and molecular areas were examined by surface activity based mass conservation plots. This shows that the peptide is practically fully accumulated in the interface. Secondary structure and orientation was analyzed by means of polarized infrared reflectivity. Brewster angle microscopy and scanning force microscopy contributed nanostructural images. At low surface pressures the molecules form anti-parallel beta-sheets lying flat on the interface. Upon a moderate increase of the lateral pressure a flat beta-turn structure appears with inter- and intramolecular H-bonds. We also observed aggregates forming fingerprint-like structures with a diameter of approximately double the hydrophobic length of a beta-turn conformation. Beyond approximately 18 mN m(-1) the beta-turns straighten up. The lowest measured tilt angle was 45 degrees at 36 mN m(-1).

Effects of heavy metals on nitrogen uptake by Paxillus involutus and mycorrhizal birch seedlings.

The effects of the heavy metals Cu, Cd, Ni, Pb and Zn on [(14)C]methylamine and [(14)C]aminoisobutyric acid uptake were studied in the free-living fungus Paxillus involutus and in mycorrhizal and non-mycorrhizal birch roots. The uptake of both N sources by P. involutus was inhibited by the five metals tested. However, Cu(2+) and Pb(2+) had a greater inhibitory effect. Non-competitive inhibitions were determined between heavy metals and [(14)C]methylamine uptake. [(14)C]Methylamine uptake was reduced by one third by 2 µM Cd(2+) and Cu(2+) in non-mycorrhizal roots, whereas that of mycorrhizal roots was not affected. However, it was reduced by 30 to 80% by 200 µM Cd(2+) and Cu(2+) irrespective of the mycorrhizal status. [(14)C]Aminoisobutyric acid uptake in mycorrhizal roots was not significantly affected by Cd(2+) and Cu(2+), whereas that of non-mycorrhizal roots was decreased by 77% at 200 µM Cu(2+). [(14)C]Aminoisobutyric acid uptake was 4.5 to 6 fold higher in mycorrhizal roots, compared with non-mycorrhizal roots, even under metal exposure. The high efficiency of N acquisition by mycorrhizal birch seedlings under metal exposure might be regarded as a mechanism of stress avoidance.

Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by ellipsometry and PM-IRRAS at the air-water interface.

The penetratin peptide, a 16 amino acid sequence extracted from Antennapedia homeodomain, is able to translocate across a neural cell membrane through an unknown mechanism, most likely a non-specific interaction with membrane lipids. Beyond its potential application as vector targeting small hydrophilic molecules and enabling them to reach a cell nucleus, this observation raises intriguing questions concerning the physico-chemistry of peptide-lipid interactions. Here we present a study of the role of lipid surface pressure and head charge on the mechanism of interaction. This was performed using optical techniques: surface infrared spectroscopy and ellipsometry, applied to a monolayer of phospholipids deposited at the air-water interface. Determination of the structure and orientation of peptides and lipids (separately or together) evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption and its action on lipids.