Effects of plant-symbiotic relationships on the living soil microbial community and microbial necromass in a long-term agro-ecosystem.

We examined the impact of arbuscular mycorrhizal fungi and rhizobia on the living microbial community and microbial necromass under different long-term fertilization treatments at the long-term Static Fertilization Experiment Bad Lauchstädt (Germany). Phospholipid fatty acids (PLFA) and amino sugars plus muramic acid, were used as biomarkers for soil microbial bio- and necromass, respectively, and analyzed from six treatments imposed on two crop rotations, varying only in the inclusion/non-inclusion of a legume. Treatments included: two levels of only farmyard manure (FYM), only mineral fertilizer (NPK), the combined application of both fertilizer types and a non-fertilized control. PLFA profiles differed clearly between the investigated crop rotations and were significantly related to labile C, mineral N, and soil pH. This emphasizes the role of carbon, and of mycorrhizal and rhizobial symbioses, as driver for changes in the microbial community composition due to effects on the living conditions in soil. We found some evidence that legume associated symbiosis with arbuscular mycorrhizal fungi and rhizobia act as a buffer, reducing the impact of varying inputs of mineral nutrients on the decomposer community. While our results support former findings that living microbial populations vary within short-term periods and are reflective of a given crop grown in a given year, soil necromass composition indicates longer term changes across the two crop rotation types, mainly shaped by fertilizer related effects on the community composition and C turnover. However, there was some evidence that specifically the presence of a legume, affects the soil necromass composition not only over the whole crop rotation but even in the short-term.

An easy method using glutaraldehyde-introduced fluorescence for the microscopic analysis of plant biotrophic interactions.

The method introduced in this article makes use of the glutaraldehyde-induced auto-fluorescence of proteins after cross-linking with glutaraldehyde for the analysis of cellular and sub-cellular structures. Because the interface of biotrophic interactions is rich in proteins, the method presented is particularly suitable for the analysis of such interactions; we have exemplified its usefulness by analyzing (1) the root feeding sites induced in roots from Arabidopsis thaliana by the root-knot nematode Meloidogyne incognita; (2) leaves from Cucurbita pepo infected by powdery mildew and (3) roots from Nicotiana tabacum colonized by the arbuscular mycorrhizal fungus Glomus intraradices. The use of confocal and multi-photon laser scanning microscopy allows three-dimensional reconstructions from optical sections of complex biotrophic interactions. In the case of root-knot nematode feeding sites, our method enabled us to simultaneously study the development of the plant xylem elements (using lignin auto-fluorescence), the nematode feeding site and the nematode itself.

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots.

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H2O2 accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl3, intracellular accumulation of H2O2 was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H2O2 was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.

A mycorrhiza-responsive protein in wheat roots.

A small protein, designated Myk15, was found to be strongly induced in wheat ( Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane alpha-helix and may correspond to a cleavable targeting domain.

Reorganization of tobacco root plastids during arbuscule development.

In the present paper we analyzed plastid populations labeled by the green fluorescent protein in non-mycorrhizal and mycorrhizal roots of tobacco (Nicotiana tahacum L.). We show by confocal laser scanning microscopy (i) a dramatic increase in these plastids in mycorrhizal roots and (ii) the formation of dense plastid networks covering the symbiotic interface of the arbuscular mycorrhiza, the arbuscule. These cytological observations point to an important role of root cortical cell plastids in the functioning of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the 'yellow pigment' and other apocarotenoids.

Plants and certain bacteria use a non-mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long-known 'yellow pigment'. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an 'apocarotenoid complex' of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.

Transfer of rps19 to the nucleus involves the gain of an RNP-binding motif which may functionally replace RPS13 in Arabidopsis mitochondria.

The discovery of disrupted rps19 genes in Arabidopsis mitochondria prompted speculation about the transfer to the nuclear compartment. We here describe the functional gene transfer of rps19 into the nucleus of Arabidopsis. Molecular cloning and sequence analysis of rps19 show that the nuclear gene encodes a long N-terminal extension. Import studies of the precursor protein indicate that only a small part of this extension is cleaved off during import. The larger part of the extension, which shows high similarity to conserved RNA-binding domains of the RNP-CS type, became part of the S19 protein. In the Escherichia coli ribosome S19 forms an RNA-binding complex as heterodimer with S13. By using immuno-analysis and import studies we show that a eubacterial-like S13 protein is absent from Arabidopsis mitochondria, and is not substituted by either a chloroplastic or a cytosolic homologue of this ribosomal protein. We therefore propose that either a highly diverged or missing RPS13 has been functionally replaced by an RNP domain that most likely derived from a glycine-rich RNA-binding protein. These results represent the first case of a functional replacement of a ribosomal protein by a common RNA-binding domain and offer a new view on the flexibility of biological systems in using well-adapted functional domains for different jobs.

Dose-response curve for chromosome translocations measured in human lymphocytes exposed to 60Co gamma rays.

Chromosome painting was employed to measure frequencies of reciprocal translocations in human lymphocytes induced by 60Co gamma rays, with emphasis on low doses. Translocations and dicentrics were distinguished by use of a pan-centromeric probe. A total of 41,151 metaphases were analyzed at doses below 0.2 Gy. The linear "take-off" slope of the linear-quadratic dose-response curve for translocations, i.e., the alpha coefficient, was measured to be 0.023 +/- 0.005 translocations per cell per Gy. This alpha coefficient is more precise than previously measured. Because the alpha coefficient is the dominant contributor to the translocation frequency induced by low-level exposures, the results presented here will substantially reduce uncertainties in biodosimetry estimates obtained for stable translocations.

Potato mitochondrial manganese superoxide dismutase is an RNA-binding protein.

An RNA-binding protein present in potato mitochondrial lysates was purified and identified as manganese-containing superoxide dismutase (MnSOD). Using a gel mobility shift assay we found that proteins from mitochondrial lysates bind with high affinity to in vitro transcripts of mitochondrial orf206, encoding a subunit of the ABC-type heme transporter. By ammonium sulfate fractionation and two subsequent chromatographic steps on MonoQ columns we purified a 28 kDa protein to apparent homogeneity. Protein sequencing identified the purified polypeptide as manganese-containing superoxide dismutase, which is a specific enzymatic scavenger of superoxides in mitochondria. Using gel mobility shift and competition assays, we show that RNA-binding of MnSOD of potato is not influenced by 400 mM KCl or heparin and is specific to heteropolymeric RNAs. The labeled mitochondrial transcript could be competed with low amounts of unlabeled transcript while binding was stable to competition with large amounts of tRNA or high concentrations of NADH and NADPH. The purified MnSOD of potato mitochondria was UV-cross-linked to the mitochondrial transcript. The Mn- and Fe-containing SODs from Escherichia coli showed no binding to the RNA by either gel mobility shift or UV-cross-linking. Enzyme activity assays revealed that binding of RNA to the mitochondrial MnSOD does not significantly influence enzyme activity. This indicates that the RNA-binding feature of MnSOD of potato mitochondria is probably not involved in modulating SOD enzyme activity and suggests a function different from superoxide degradation as ist biological role.

RNA editing in higher plant mitochondria: analysis of biochemistry and specificity.

RNA editing alters genomically encoded cytidines to uridines posttranscriptionally in higher plant mitochondria. Most of these editing events occur in translated regions and consequently alter the amino acid sequence. In Oenothera berteriana more than 500 editing sites have been detected and the total number of editing sites exceeds 1000 sites in this mitochondrial genome. To identify the components involved in this process we investigated the factors determining the specificity of RNA editing and the apparent conversion of cytidine to uridine residues. The possible biochemical reactions responsible for RNA editing in plant mitochondria are de- or transamination, base substitution and nucleotide replacement. In order to discriminate between these different biochemical mechanisms we followed the fate of the sugar-phosphate backbone by analysing radiolabeled nucleotides after incorporation into high molecular mass RNA. Plant mitochondria were supplied with [alpha-32P]CTP to radiolabel CMP residues in newly synthesized transcripts. Radiolabeled mtRNA was extracted and digested with nuclease P1 to hydrolyse the RNA to monophosphates. The resulting monophosphates were analysed on one- and two-dimensional TLC systems to separate pC from pU. Radiolabeled pU was detected in increasing quantities during the course of incubation. These results suggest that RNA editing in plant mitochondria involves either a deamination or a transglycosylation reaction. The editing product was identified as uridine and not as a hypermodified nucleotide which is recognized as uridine. Similar results have been obtained by incubating in vitro transcribed mRNAs with mitochondrial lysates indicating that RNA editing and transcription is not directly linked in plant mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)