Janine Brouillette

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid.

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after 13C1 glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using 13C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.

Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase

A tonoplast-type vesicle preparation, substantially free from other subcellular membranes, was obtained from corn roots by equilibrium sucrose density gradient centrifugation. At pH 6.5 and in the presence of chloride ions, the tonoplast-type ATPase activity as measured by Pi release, was inhibited by nitrate ions. The ATPase activity was insensitive to molybdate and vanadate, indicating a minimum nonspecific phosphatase and plasma membrane contamination. The vesicles exhibited an ATP hydrolysis-supported proton uptake which was measured by the absorption change of acridine orange. The ATP hydrolysis supported uptake and the subsequent perturbant-induced release of protons (decay) was described by a kinetic model which was previously developed to evaluate the coupling between proton pumping and the primary energy yielding process for other biomembranes. The proton pumping activity was more sensitive to nitrate ions then was ATP hydrolysis. The differential effect and the kinetic analysis of nitrate inhibition led us to suggest that (i) the coupling between Pi release and proton pumping was indirect in nature and (ii) the primary inhibitory effect of nitrate ion was originated from an interaction with a protogenic protein domain which is functionally linked to the ATPase in the tonoplast-type membrane.

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.

THE STRUCTURE OF THE EXOPOLYSACCHARIDE OF PSEUDOMONAS FLUORESCENS STRAIN H13

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

Cyclomaltoheptaose (β-cyclodextrin) and hydroxyethyl-substituted β-cyclodextrin inclusion complex formation with chlorogenic acid: solvent effects on inclusion complex stability

Abstract The inclusion complexes of cyclomaltoheptaose (β-CD) and β-CDs 50% hydroxyethyl-substituted derivative (HE-β-CD) with chlorogenic acid (CA) were studied with regard to temperature and water activity (aH2O ≈ mole fraction = XH2O = 0.8–0.99; 0.1 M Na phosphate buffer) utilizing first-derivative spectrophotometric analyses of bathochromic shifts (Δλ) in CAs UV absorbance as a function of variable [CD]. From the dependence of the apparent stability constant, K, on X H 2 O (K = K ‡ X H 2 O z ) we estimated that the β-CD · CA complexs apparent stoichiometric coefficient, z, for water was ca. 7 ± 1 (K ‡ = 1032 ± 54 M −1 ) ; this value agrees with recently published literature concerning the minimum number of waters needed to stabilize a similar β-CD adduct. However, we determined that z was significantly lower ( 4 ± 0.3; K ‡ = 809 ± 31 M − ) for the HE-β-CD · CA complex. These results argue that a unique species of bound water is involved in β-CD · CA stability since a 50% substitution resulted in an equivalent loss in z as well as substantial decrease in K ‡ . This hypothesis was supported by NMR inversion recovery experiments whereupon the most significant perturbation to spin-lattice relaxation (ΔT1 = T1β-CD − T1β-CD · CA) was associated with β-CDs 1H at position 3 (H−3; ΔT1 = 585 ms). Small ΔT1s were also observed for H-2 (160 ms) and H-6,6′ (83 ms). β-CDs ΔT1s were dependent not only upon the adducts concentration but also diminished at a high ionic strength. These data indicate that ΔT1 was related to changes in [D2O] at or near β-CDs hydroxyl groups and that these D2O molecules were bound with a relatively long residence time. Thermochemical measurements of ΔH and ΔS at various XH2Os display typically linear enthalpy-entropy compensation (ΔH−ΔS) relationships but with a slope (Tc = ∂ΔH/∂ΔS = 272 K) significantly less than standard aqueous thermodynamic measurements (Tc = 305 K) of a similar system. This unequivocal XH2O effect on Tc argues that the chemical part process of CD · guest adduct formation involves changes in relative solvation, presumably desolvation, of β-CDs binding site. This interpretation was supported by the dependency of Δλmax on CD binding site dimension and XH2OMeOH.