Structural analysis of a plant sucrose carrier using monoclonal antibodies and bacteriophage lambda surface display.
Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane. (+info)
Soft gel medium solidified with gellan gum for preliminary screening for root-associating, free-living nitrogen-fixing bacteria inhabiting the rhizoplane of plants.
For preliminary screening for and characterization of free-living nitrogen-fixing bacteria from rhizoplane microflora, we used Winogradsky's mineral mixture-based nitrogen-free medium solidified with 0.3% gellan gum. The soft gel medium enabled some reference and wild free-living nitrogen-fixing bacteria to grow in characteristic colonies, including their reaction to oxygen and their motility change. Gellan gum is thus likely to be a better gel matrix than agarose for the investigation of root-associating, free-living nitrogen-fixing bacteria to identify their characteristic behaviors. (+info)
Negative feedback within a mutualism: host-specific growth of mycorrhizal fungi reduces plant benefit.
A basic tenet of ecology is that negative feedback on abundance plays an important part in the coexistence of species within guilds. Mutualistic interactions generate positive feedbacks on abundance and therefore are not thought to contribute to the maintenance of diversity. Here, I report evidence of negative feedback on plant growth through changes in the composition of their mutualistic fungal symbionts, arbuscular mycorrhizal (AM) fungi. Negative feedback results from asymmetries in the delivery of benefit between plant and AM fungal species in which the AM fungus that grows best with the plant Plantago lanceolata is a poor growth promoter for Plantago. Growth of Plantago is, instead, best promoted by the AM fungal species that accumulate with a second plant species, Panicum sphaerocarpon. The resulting community dynamic leads to a decline in mutualistic benefit received by Plantago, and can contribute to the coexistence of these two competing plant species. (+info)
Cloning and expression of biologically active Plantago lanceolata pollen allergen Pla l 1 in the yeast Pichia pastoris.
The glycoprotein Pla l 1 is the major allergen from English plantain (Plantago lanceolata) pollen, which is a common cause of pollinosis in temperate areas. Three complete cDNAs for Pla l 1 isoforms were isolated by PCR using specific 3' and 5' primers. All three Pla l 1 cDNAs code for a 25-residue leader peptide and a 131-residue mature protein that contains two polymorphic positions, an N-glycosylation site at position 107 and six cysteine residues involved in three disulphide bridges. The allergen variant Pla l 1.0101 was produced in Pichia pastoris at a yield of 20 mg per litre of culture as a mixture of non-glycosylated (17 kDa), glycosylated (23 kDa) and dimeric (32-39 kDa) forms. Recombinant Pla l 1 (rPla l 1) was purified by affinity chromatography with an anti-natural Pla l 1 (anti-nPla l 1) monoclonal antibody, and its molecular and immunological properties were compared with the natural allergen by CD spectroscopic analysis, enzymic deglycosylation, lectin-binding assay, immunodetection and ELISA-inhibition assays using sera from plantain-allergic patients. The recombinant allergen is properly folded, as deduced from CD spectra, and the immunodominant allergenic epitopes of the natural allergen are preserved in rPla l 1. These results allow us to conclude that P. pastoris is a convenient system for the efficient production of biologically active rPla l 1, which could have a potential use for clinical purposes. Furthermore, a sequence similarity of Pla l 1 to the major allergen from the olive tree pollen, Ole e 1, is revealed in this work, and the allergenic cross-reactivity between both allergens has been studied. (+info)
Rapid turnover of hyphae of mycorrhizal fungi determined by AMS microanalysis of 14C.
Processes in the soil remain among the least well-characterized components of the carbon cycle. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts in many terrestrial ecosystems and account for a large fraction of photosynthate in a wide range of ecosystems; they therefore play a key role in the terrestrial carbon cycle. A large part of the fungal mycelium is outside the root (the extraradical mycelium, ERM) and, because of the dispersed growth pattern and the small diameter of the hyphae (<5 micrometers), exceptionally difficult to study quantitatively. Critically, the longevity of these fine hyphae has never been measured, although it is assumed to be short. To quantify carbon turnover in these hyphae, we exposed mycorrhizal plants to fossil ("carbon-14-dead") carbon dioxide and collected samples of ERM hyphae (up to 116 micrograms) over the following 29 days. Analyses of their carbon-14 content by accelerator mass spectrometry (AMS) showed that most ERM hyphae of AM fungi live, on average, 5 to 6 days. This high turnover rate reveals a large and rapid mycorrhizal pathway of carbon in the soil carbon cycle. (+info)
PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major.
Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters. For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias. For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions. Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family. The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose. In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago. Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues. Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed. (+info)
Psyllium husk. I: Effect on plasma lipoproteins, cholesterol metabolism, and atherosclerosis in African green monkeys.
Psyllium's effects on plasma and lipoprotein cholesterol concentrations, cholesterol metabolism, and diet-induced atherosclerosis were studied in adult male African green monkeys (Cercopithecus aethiops). Animals were fed for 3.5 y one of three experimental diets: low-cholesterol cellulose (LCC), high-cholesterol cellulose (HCC), or high-cholesterol psyllium (HCP). The LCC and HCP groups had significantly (P less than 0.05) lower plasma cholesterol concentrations (39% lower) at 1 mo than did the HCC group. These responses persisted throughout the study. Plasma cholesterol changes were due to a reduction in intermediate-density and low-density lipoproteins; very-low and high-density-lipoprotein concentrations were similar among groups. Aortic atherosclerosis, evaluated as percent sudanophilia at 3.5 y, was lowest in the LCC group, intermediate in the HCP group, and highest in the HCC group. Cholesterol absorption, neutral steroid and fat excretion, HMGCoA reductase activity (in intestine and liver), and body weight were unrelated to psyllium's hypocholesterolemic effects. (+info)
Psyllium husk. II: Effect on the metabolism of apolipoprotein B in African green monkeys.
Dietary psyllium's ability to reduce low-density-lipoprotein (LDL) cholesterol is presumably mediated by increased LDL catabolism and/or reduced LDL synthesis. To distinguish between these possibilities, apolipoprotein B (apo B) metabolism was studied in adult male African green monkeys consuming one of three semipurified diets: low-cholesterol cellulose (LCC), high-cholesterol cellulose (HCC), or high-cholesterol psyllium (HCP). 131I-labeled LDL and 125I-labeled VLDL were injected simultaneously into animals; blood samples were drawn at selected times and apo B specific activity determined in VLDL, IDL, and LDL. Based on a multicompartmental model, LDL apo B pool size and de novo apo B transport were elevated significantly in HCC animals compared with HCP and LCC animals. Differences in LDL transport, although not significant, paralleled differences observed in LDL apo B pool size. Fractional catabolic rates were similar among groups (HCC 0.040 +/- 0.010; HCP 0.042 +/- 0.009, and LCC 0.043 +/- 0.004 pools/h). These data suggest that dietary psyllium reduces plasma cholesterol concentrations by decreasing LDL synthesis. (+info)