Core Rot Development in Red Delicious Apples Is Affected by Susceptibility of the Seed Locule to Alternaria alternata Colonization.

ABSTRACT Alternaria alternata is the causal organism of core rot decay symptoms in susceptible cv. Red Delicious but not in resistant cv. Golden Delicious. The two cultivars did not differ in natural colonization of the style and ovary during the first week after full bloom; colonization of the ovary in the susceptible cultivar subsequently decreased with increasing distance from the calycine tube. By 30 days after full bloom, Alternaria recovery from ovary 1, adjacent to the end of the calycine tube, was 100 and 40% in the susceptible and resistant cultivars, respectively. In the susceptible cultivar, Alternaria recovery decreased from 75 to 20% in ovaries 2, 3, and 4, while there was only minor incidence in the resistant cultivar. Inoculation of the mesoderms of the two cultivars induced similar decay symptoms, but inoculated locules of Red Delicious were more susceptible than those of Golden Delicious. Increased inoculum concentration or isolate virulence enhanced the difference in locule susceptibility between the cultivars. Inoculation on isolated seed locules or on media amended with susceptible locule tissue as a carbon source induced greater transcript levels of several genes than the inoculation on resistant tissue. Endo- and exoglucanase activity levels were higher at pH 4.8 than at 4.2, conditions typical of the mesoderm adjacent to the seed locules of the susceptible and resistant cultivars, respectively. Current results suggest that susceptibility of Red Delicious apples to core rot decay is dependent on the sensitivity to locule colonization and on mesoderm pH, a factor that enhances fungal virulence.

Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells.

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.

Control of Moldy-Core Decay in Apple Fruits by ß-Aminobutyric Acids and Potassium Phosphites.

Alternaria alternata is the predominant fungal pathogen responsible for moldy-core decay in the 'Red Delicious' apple fruits. The failure of registered fungicides to control the disease necessitated the search for alternative methods. Here we report that DL-ß-aminobutyric acids (BABA) and potassium phosphite are capable of controlling moldy-core in apple fruits in the laboratory and the field. Laboratory tests involving inoculation of wounded mature fruits revealed that decay by A. alternata was 40 to 58% inhibited by 50 µg/ml and completely inhibited by 500 µg/ml of potassium phosphite. DL-BABA, R-BABA, and S-BABA at a concentration of 500 µg/ml inhibited decay formation by 82 to 90%. Potassium phosphite, BABA, and S-BABA inhibited decay formation even when applied 6 to 48 h postinoculation. In vitro tests indicated that BABA compounds did not affect conidial germination or mycelial growth, whereas potassium phosphite partially inhibited both developmental stages of the fungus. Results suggest that BABA inhibits fruit decay development indirectly, probably by inducing resistance in the host tissue, whereas potassium phosphite may act directly against the fungus. A preliminary field trial in a commercial apple orchard showed that three foliar applications of BABA or potassium phosphite, starting from the beginning of bloom until petal fall, reduced the number of fruits infected with moldy-core by 40 and 60%, respectively, relative to fruits from nontreated control trees. Results suggest that potassium phosphite and BABA could provide adequate control of moldy-core disease in apple.

Proline transport into brush border membrane vesicles from the midgut of Manduca sexta larvae.

The unidirectional transport of proline by midgut epithelium cells of the tobacco hornworm, Manduca sexta, was investigated in brush border membrane vesicles. Both K.(+)-stimulated and K(+)-insensitive transport pathways were identified. Analyses of K(+)-dependent proline transport revealed 1:1 ratio of K+ to proline, a Km of 13 mM for K+ and a decrease in both Km (from 18 mM to 3 mM) and Vmax (from 37 nmol/mg protein/min to 10 nmol/mg protein/min) for proline in the presence of a K+ gradient. The profiles of cis-inhibition by other amino acids demonstrated that proline is transported into midgut cells by a transport system that is shared by other neutral amino acids.

Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.

Separation of cytochrome P-450 containing vesicles from the midgut microsomal fraction of Manduca sexta.

1. Microsomes prepared from midgut tissue of Manduca sexta by differential centrifugation are a heterogeneous population of vesicles. 2. Upon centrifugation of microsomes in a sucrose gradient of 30%, 15% and 10%, specific activity of cytochrome P-450 catalyzed O-demethylation of p-nitroanisole (a marker enzyme for endoplasmic reticulum) was increased 2.5 to 3.5-fold in the 15% fraction. 3. Specific activities of alkaline phosphatase and leucine aminopeptidase (marker enzymes for brush border membranes) were increased 3.1 to 5.7-fold in the pellet. 4. Differential centrifugation is a useful step in the purification of cytochrome P-450 enzymes.

Differential inhibition by Bacillus thuringiensis delta endotoxin of leucine and aspartic acid uptake into BBMV from midgut of Manduca sexta.

Pre-incubation of brush border membrane vesicles (BBMV) isolated from the midgut of Manduca sexta with activated Bacillus thuringiensis delta endotoxin for a short period resulted in differential inhibition of K(+)-dependent transport of leucine relative to the effect on K(+)-dependent transport of aspartic acid. The difference in I1/2 (5 fold greater for aspartic acid than for leucine) is interpreted as the result of enhanced binding of the B. thuringiensis delta endotoxin to the leucine transport system.

Enhanced Net K Uptake Capacity of NaCl-Adapted Cells.

Maintenance of intracellular K(+) concentrations that are not growth-limiting, in an environment of high Na(+), is characteristic of NaCl-adapted cells of the glycophyte, tobacco (Nicotiana tabacum/gossii). These cells exhibited a substantially greater uptake of (86)Rb(+) (i.e. an indicator of K(+)) relative to unadapted cells. Potassium uptake into NaCl-adapted cells was 1.5-fold greater than unadapted cells at 0 NaCl and 3.5-fold greater when cells were exposed to 160 millimolar NaCl. The difference in net K(+) uptake between unadapted and NaCl-adapted cells was due primarily to higher rates of entry rather than to reduced K(+) leakage. Presumably, enhanced K(+) uptake into adapted cells is a result of electrophoretic flux, and a component of uptake may be linked to vanadate-sensitive H(+) extrusion.

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation.

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H(+)-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO(3) (-) sensitive H(+) transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H(+) transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H(+) transport capacity after adaptation was also observed for ATP hydrolysis.

Osmotically induced proton extrusion from carrot cells in suspension culture.

Addition of 200 mm of a polyol to anthocyanin containing carrot (Daucus carota L.) cells in suspension culture decreased turgor pressure to zero and induced hyperpolarization of the membrane potential and acidification of the medium due to H(+) extrusion. These changes were shown to be slightly affected by vanadate. In parallel, a decrease in intracellular ATP and total adenylate concentrations were observed. However, when the osmoticum was NaCl acidification of the medium occurred in the absence of considerable changes in intracellular ATP concentration. These results are interpreted as indicating that a drop of turgor, by addition of a polyol, triggers a proton extrusion activity which is only slightly inhibited by vanadate but apparently ATP utilizing. The observed decrease in ATP level occurs without a change in respiration rate and is accompanied by a drop in total adenylate pool. However when NaCl is the osmoticum it is assumed that Delta(muH+) is enhanced through a Na(+)/H(+) antiporter. The difference between the two types of osmotica as related to their ability to penetrate through the cellular membrane is discussed.

Changes in Membrane Potential as a Demonstration of Selective Pore Formation in the Plasmalemma by Poly-l-Lysine Treatment.

A technique which allows determination of solute pool concentrations in the cytosol was developed exploiting the interaction between a polycation and the anionic sites of the plasmalemma. It was shown that treatment of Nicotiana tabacum, cv Xanthi, cells in suspension culture with an appropriate concentration of poly-l-lysine induced pore formation selectively in the plasmalemma. The data presented in this paper shows that the plasmalemma of all the cells was affected while the tonoplast remained undamaged. This conclusion is based on the facts that treatment of the cells with the minimum amount of poly-l-lysine which just abolishes the electrogenic potential (similarly to carbonyl cyanide-p-trifluormethoxyphenylhydrazone and NaN(3)) induces the leakage of only a small fraction of the K(+) present in the cells. These effects of poly-l-lysine differ from the effects of polymyxin B which induces total leakage of low molecular weight solutes (R. Weimberg, H. R. Lerner, A. Poljakoff-Mayber 1983 J Exp Bot 34: 1333-1346) and therefore affects also the tonoplast.Membrane potential was determined using the partition of the lipophilic cation tetraphenylphosphonium. The electrogenic component of the membrane potential was estimated using carbonyl cyanide-p-trifluormethoxyphenylhydrazone and azide. Poly-l-lysine treatment was used to measure K(+) compartmentation in Nicotiana cells grown in a NaCl-containing medium.