Nitrogen and carbon/nitrogen dynamics in arbuscular mycorrhiza: the great unknown.

Many studies have established that arbuscular mycorrhizal fungi transfer N to the host plant. However, the role and importance of arbuscular mycorrhiza (AM) in plant N nutrition is still uncertain, as are the C/N interactions within the symbiosis. Published reports provide differing, and often contradictory, results that are difficult to combine in a coherent framework. This review explores questions such as: What makes the difference between a positive and a negative effect of AM on plant N nutrition? Is the mycorrhizal N response (MNR) correlated to the mycorrhizal growth response (MGR), and how or under which conditions? Is the MNR effect on plant growth C mediated? Is plant C investment on fungal growth related to N needs or N benefit? How is the N for C trade between symbionts regulated? The patternless nature of current knowledge is made evident, and possible reasons for this are discussed.

Defense related phytohormones regulation in arbuscular mycorrhizal symbioses depends on the partner genotypes.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Modulation of the hormonal and transcriptional profiles, including changes related to defense signalling, has been reported in many host plants during AM symbioses. These changes have been often related to the improved stress tolerance common in mycorrhizal plants. However, results on the alterations in phytohormones content and their role on the symbiosis are controversial. Here, an integrative analysis of the response of phylogenetically diverse plants (i.e., tomato, soybean, and maize) to two mycorrhizal fungi -Funneliformis mosseae and Rhizophagus irregularis- was performed. The analysis of the defense-related hormones salicylic acid, abscisic acid, and jasmonates, and the expression of marker genes of the pathways they regulate, revealed significant changes in the roots of mycorrhizal plants. These changes depended on both the plant and the AM fungus (AMF) involved. However, general trends can be identified: roots associated with the most effective colonizer R. irregularis showed fewer changes in these defense-related traits, while the colonization by F. mosseae led to significant modifications in all plants tested. The up-regulation of the jasmonate pathway by F. mosseae was found to be highly conserved among the different plant species, suggesting an important role of jasmonates during this AM interaction. Our study evidences a strong influence of the AMF genotype on the modulation of host defense signalling, and offers hints on the role of these changes in the symbiosis.

Kinetics of NH (4) (+) uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25 mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100 µM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1 mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

GintMT1 encodes a functional metallothionein in Glomus intraradices that responds to oxidative stress.

A full-length metallothionein (MT) gene (GintMT1) was isolated from Glomus intraradices extraradical mycelium. This is the first MT gene reported in the genus Glomus, third in the Glomeromycota. Functional analysis of GintMT1 in a MT-defective Saccharomyces cerevisiae strain indicates that it encodes a functional MT. Gene expression analyses revealed that the transcript levels of GintMT1 were elevated in mycelia treated with 5 mM Cu or paraquat but inhibited in mycelia treated with 50 microM Cu or 450 microM Cd. The elevated expression of GintMT1 in the 5 mM Cu-treated mycelia together with the ability of GintMT1 to provide tolerance to a Cu-sensitive yeast suggests that GintMT1 might afford protection against Cu. Induction of GintMT1 expression by paraquat and 5 mM Cu, treatments that also produced an oxidative damage to the fungal membranes, suggests that GintMT1 may play a role in the regulation of the redox status of the extraradical mycelium of G. intraradices.

Expression of a tomato sugar transporter is increased in leaves of mycorrhizal or Phytophthora parasitica-infected plants.

A full-length cDNA clone (LeST3), encoding a putative tomato sugar transporter, was isolated from mycorrhizal roots by using a PCR-based approach. Based on sequence similarity, conserved motifs and predicted membrane topology, LeST3 was classified as a putative monosaccharide transporter of the sugar transporter subgroup of the major facilitator superfamily. Southern blot analysis showed that LeST3 represents a single-copy gene in tomato. To investigate its function, LeST3 was expressed in a hexose transport-deficient mutant of Saccharomyces cerevisiae. Although LeST3 was correctly transcribed in yeast, it did not restore growth on hexoses of the S. cerevisiae mutant. LeST3 gene expression was increased in the leaves of plants colonised by the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices and in those of plants infected with the root pathogen Phytophthora parasitica. These data suggest that LeST3 plays a role in the transport of sugars into the sink tissues and responds to the increased demand for carbohydrates exerted by two AM fungi and by a root pathogen to cope with the increased metabolic activity of the colonised/infected tissues or to supply carbohydrates to the AM fungus.

Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza.

Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H(+)-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.

The plasma membrane H(+)-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae.

To identify genes that encode plasma membrane H(+)-ATPases in the arbuscular mycorrhizal fungus Glomus mosseae two sets of degenerate primers matching highly conserved motifs present in all plant and fungal ATPases were designed. Nested PCR-amplification of G. mosseae genomic DNA using the designed degenerate primers was carried out. Sequence analysis of the cloned PCR products identified five different clones (GmHA1, GmHA2, GmHA3, GmHA4 and GmHA5) encoding putative plasma membrane H(+)-ATPases. Comparison of the deduced amino-acid sequences of GmHA1-GmHA5 indicate that GmHA1, GmHA3 and GmHA4 are highly identical, while GmHA2 and GmHA5 are more divergent. The evolutionary and functional significance of the divergence found among the different members of the H(+)-ATPase gene family in G. mosseae is discussed.

A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato.

Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon Ca2+-ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots.

Neurogenic cells inhibit the differentiation of cardiogenic cells.

In this study, we present evidence that neurogenic cells inhibit the differentiation of cardiogenic cells. When cells of the entire area pellucida at stage 5 were dissociated and reaggregated, the aggregates differentiated into neural tissues and other structures of any germ layer origin, except for heart tissues, despite the fact that the cardiogenic cells are already committed to differentiate. The phenomenon also occurs from stages 6 to 8, during which cells are in a higher state of commitment. Using combinations of different regions of the area pellucida, it was confirmed that neurogenic cells are responsible for the inhibition of cardiogenic cell differentiation. The inhibition is not species-specific because quail and chick neurogenic cells inhibit each other's cardiogenic cell differentiation. Direct contact between cardiogenic and neurogenic cells seems to be necessary for inhibition because cardiogenic cell differentiation takes place if cardiogenic and neurogenic cells are separated by a porous membrane. Dissociated neural tubes from early stages, but not dissociated telencephalon from 2-day-old chicks, also inhibit cardiogenic cell differentiation. These results suggest that neurogenic cells may sequester molecules necessary for the differentiation of cardiogenic cells rather than produce inhibitory molecules.

Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes.

Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.

Early in-vitro histological chondral differentiation.

In vitro chondrogenesis is possible in the chick embryo from stage 4 of Hamburger and Hamilton (1951), only 18-19 hours of incubation, before somite formation. In stage 4 of Hamburger and Hamilton (1951) the chondroblasts are placed laterally to the primitive streak and notochord cells are not necessary for cartilage differentiation.

Characterization of proton extrusion in sunflower cell cultures.

Cell suspensions derived from callus root tips of sunflower (Helianthus annuus L., cv. enano) were obtained in order to assess the effects of different chemical and physical agents on cell H+ extrusion. Cell H+ efflux was sensitive to temperature, pH, inhibitors of plasmalemma H(+)-ATPase and Ca2+ and K+ concentrations in the assay medium, as well as to the light intensity at which cells were cultivated. Thus, in the darkness and at 60 mumol/m2/s of illumination, a strong inhibition of H+ extrusion was detected as compared to cells grown at 30 mumol/m2/s. H+ extrusion by cells grown at 30 mumol/m2/s was unaffected by the presence of calcium in the assay medium, while at 60 mumol/m2/s such an activity increased when calcium was removed. These results provide the basis for the use of cell suspensions as an appropriate model to investigate the involvement of membrane-associated processes in plant tolerance mechanisms to different environmental stresses.

Effects of white light on the pineal gland of the chick embryo.

Chick embryos were directly exposed to a source of white light during incubation and sacrificed before hatching. The light caused a number of teratological effects such as high mortality, delay in development, celosomy, hepatomegaly, auricular dilation and micrognatia. The pineal gland of the illuminated embryos showed an increase in number and size of the intracytoplasmic lipid droplets of the follicular pinealocytes. These findings suggest that the pineal gland of the chick embryo is sensitive to light.

Diencephalic origin of the pineal gland of the chicken embryo.

In the present paper, the diencephalic origin of the chick pineal gland was analyzed by a series of experiments: prosencephalic substitution; in vitro culture of isolated diencephalons; and total or partial excission of the diencephalic roof. The results indicate that the differentiation of the chick pineal gland in the rooof of the third ventricle is not influenced by the neighbouring brain vesicles and is of diencephalic origin. Moreover, in order to obtain chick embryos with pineal agenesia, the whole diencephalic roof has to be removed.

Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen.

Proteins, acyl-lipids and polysaccharides from cell walls of mature and germinated olive pollen were studied. In general, hemicelluloses are the most abundant polysaccharides, arabinose in mature and glucose in germinated pollen being the main components of these macromolecules. Protein content and its amino acid composition are very similar in walls from mature and germinated pollen, these compounds showing a weak acid character. Free-fatty acids are the most abundant lipid molecules in mature and germinated pollen walls and a decrease in acyl-lipids, especially in polar lipids, as well as a higher unsaturation of their fatty acid components are observed after germination.

[Neural differentiation starting with very young dissociated cells]. / Différenciation neurale à partir de cellules très jeunes dissociées

Before the appearance of the morphological primordium of the nervous dissociated, reaggregate selectively "in vitro" and reach high levels of neural differentiation and organization. This property allows the experimental reproduction of the neural differentiation process. Before the appearance of the morphological primordium of the nervous system, its precursory cells carry the necessary information to put in action the basic characteristics of cell differentiation and structural organization, even isolated from the influence of mesoblastic cells.