On-farm production of AM fungus inoculum in mixtures of compost and vermiculite.

On-farm production of arbuscular mycorrhizal (AM) fungus inoculum can reduce the cost of the inoculum and increase utilization of this symbiosis in plant production. Bahiagrass (Paspalum notatum Flugge) seedlings, colonized by AM fungi, were transplanted into raised bed enclosures. Media within the enclosures was vermiculite mixed with either field soil or yard clippings compost in Experiment I and vermiculite mixed with yard clippings compost or dairy manure/leaf compost in Experiment II. Compost and vermiculite mixtures yielded more propagules of AM fungi than soil-based mixtures in Experiment I. Growth of plants in a 1:4 (v/v) mixture of yard clippings compost and vermiculite produced more inoculum (503 propagules cm(-3)) than growth in 1:9 and 1:99 (v/v) mixtures (240 and 42 propagules cm(-3), respectively). Water, inorganic nutrient solution minus P, and fish protein digest were added to inoculum production enclosures in Experiment II. Results indicated that supplemental nutrient addition was unnecessary. This method produces a concentrated inoculum of AM fungi in a form readily used as an amendment to horticultural potting media for the production of vegetable seedlings.

The differential effects of cell wall-associated phenolics, cell walls, and cytosolic phenolics of host and non-host roots on the growth of two species of AM fungi.

Experiments were conducted to test the hypothesis that cellular compounds, especially wall-associated compounds, released during emergence of secondary roots, stimulate the growth of arbuscular mycorrhizal (AM) fungi. Purified cell walls, crude cell-wall extracts, crude cytoplasmic extracts, and phenolic compounds previously identified as cell wall-associated, from Ri T-DNA-transformed roots of host (Daucus carota L.) and non-host (Beta vulgaris L.) were incorporated into growth medium and tested for their effects upon growth of the AM fungi Gigaspora gigantea (Nicol. & Gerd.) Gerdemann and Trappe and Gigaspora margarita Becker and Hall. Purified cell walls of both plants had little effect on G. gigantea but non-host cell walls inhibited the growth of G. margarita. Ferulic acid, a major constituent of non-host root, depressed the growth of both fungi. Nothing tested which was unique to the non-host root affected hyphal growth to the point that contact would be prohibited. Caffeic acid, found in D. carota cytoplasm, also depressed growth of both fungi. Para-hydroxybenzoic acid, a constituent of D. carota roots, stimulated growth of G. margarita hyphae, but did not affect hyphal growth of G. gigantea. Vanillic acid, unique to D. carota root cell-wall extracts, stimulated hyphal growth and branching of both fungi, and should increase the probability of contact between fungus and host root.

Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase.

The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs.

Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava.

Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel- mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs.

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with beta-glucosidase, beta-GIcNAcase, and alpha-mannosidase, respectively. Swainsonine specifically inhibited alpha-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited beta-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans.

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.

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.

Effects of multivalent cations on cell wall-associated Acid phosphatase activity.

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu(2+), Mg(2+), Zn(2+), and Mn(2+); unaffected by Ba(2+), Cd(2+), and Pb(2+); and inhibited by Al(3+). The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg(2+). On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al(3+) and Hg(2+) was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg(2+). In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca(2+) significantly reduced the effects of Hg(2+) or Al(3+), but not Mg(2+), to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg(2+) or Al(3+) which caused a Ca(2+)-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.

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.

beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation.

Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that beta-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the beta-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.

Regulatory effects of adenosine diphosphate on the activity of the plasma membrane ATPase of corn roots.

Plasma membrane enriched microsomal fraction was isolated from corn root cells by sucrose density centrifugation. The ATPase activity as measured by the release rate of inorganic phosphate, was decreased by the presence of modifiers which included diethylstilbestrol, vanadate, N,N'-dicyclohexylcarbodiimide, and miconazole. The presence of ADP also decreased the rate of ATP hydrolysis. Furthermore, a preincubation of the membrane with ADP significantly reduced the inhibitory effects of these membrane ATPase modifiers. Since the modes of interaction of these modifiers with the enzyme are different, the results suggest that the binding of ADP may stabilize the plasma membrane ATPase in a modifier insensitive state.

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain.

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K(+)-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.

Effect of lanthanum on ion absorption in corn roots.

Short term (10 min) influx of (86)Rb-labeled potassium into corn (Zea mays L. WF9 x M14) root segments was inhibited by La (NO(3))(3) or LaCl(3). Half maximal inhibition of K(+) influx from 0.25 mm KCl was obtained with 0.025 mm La(3+). Kinetic analysis indicated the inhibition to be of a competitive nature. With absorption periods exceeding one hour, La(3+) no longer inhibited, but rather stimulated K(+) influx rates. La(3+) was not an inhibitor of (36)Cl or (32)P absorption. Separated cortex and stele absorbed labeled potassium (and phosphate) at comparable rates, and La(3+) inhibited potassium influx in both tissues. The effects of La(3+) on ion absorption were similar to those of Ca(2+), suggesting that the two polyvalent cations act at the same site. Based on this and the observation that La(3+) does not seem to penetrate the plasma membrane, it was concluded that La(3+) and Ca(2+) affect changes in ion transport without entering cells.

The casparian strip as a barrier to the movement of lanthanum in corn roots.

The effectiveness of the Casparian strip as a barrier to apoplastic movement of solutes from cortex to stele of corn roots was investigated by using lanthanum in combination with electron microscopy. Lanthanum deposits were found only in cell walls and on the outside of the plasma membrane of epidermal, cortical, and endodermal cells up to the Casparian strip. Lanthanum was completely absent from the stele, indicating that the Casparian strip provides an effective barrier to apoplastic movement of solutes. Inhibitory effects of trivalent lanthanum ions on the absorption of potassium ions are discussed in relation to the nature of the lanthanum ion binding site on membranes.