PMC

Effect of increasing concentrations of the detergent SDS on V_m. Lower concentrations (from 50–100 μg mL⁻¹) had no effect, whereas at higher concentration (500 μg mL⁻¹) a considerable V_m depolarization was observed, even after washing with fresh buffer (double arrow). The single arrow indicates start of perfusion after V_m stabilization. Metric bars = sd.

Effect of S. littoralis oral secretions and regurgitants (R) on the V_m of Lima bean palisade cells. At the lowest concentration(100 μg mL⁻¹) R caused an intermediate V_m depolarization when compared to concentrations of 250 and 300 μg mL⁻¹. After washing the tissues with fresh buffer (double arrow) V_m was hyperpolarized at all concentrations, and once again R at 100 μg mL⁻¹ had an intermediate value. The single arrow indicates start of perfusion after V_m stabilization. Metric bars = sd.

Lima bean leaf V_m values as a function of distance from the bite zone 15 min after herbivore damage. The histogram superimposed on Lima bean leaf wounded by a larva of S. littoralis represents V_m values (and sd) measured at increasing distances from the bite zone. The dotted line (and its sd) represents the average V_m value from a mechanically wounded Lima bean leaf. In the close vicinity of the bite zone (up to 1.5 mm) there is a strong drop in the V_m (depolarization), whereas at about 2.5 to 3 mm from the bite zone an increase of V_m is observed (hyperpolarization). About 6 mm from the bite zone throughout all leaf there is a constant V_m depolarization.

A, V_m changes after perfusion of Lima bean leaves with 17-R,S-volicitin (= N-[17R,S-hydroxylinoleoyl]-l-Gln) and the naturally occurring 17S-volicitin (= N-[17S-hydroxylinoleoyl]-l-Gln). At all concentrations, both the racemic mixture and the natural volicitin caused no variation of V_m. B, Effect on the V_m of different N-palmitoleoyl-l-Gln concentrations. The highest concentration (300 μg mL⁻¹) had lower V_m hyperpolarization effects than 100 μg mL⁻¹, whereas at 25 μg mL⁻¹ N-palmitoleoyl-l-Gln caused a constant V_m depolarization even after washing with fresh buffer (double arrow). C, Effect of different N-linolenoyl-l-Gln concentrations on V_m. Clear effects were only caused by concentrations at 100 μg mL⁻¹. A single arrow indicates start of perfusion after V_m stabilization. The double arrow indicates washing with fresh buffer. Metric bars = sd.

Percentage of Fluo-3 AM fluorescence (y axis) as a function of the distance (in μm; x axis) from the bite zone. The experiment was designed in order to evaluate the effect of the presence or absence of exogenous Ca²⁺ in the incubation media and the presence or absence of the Ca²⁺ channel blocker Verapamil. Mechanical wounding was inferred with a razor blade. Values are the mean of at least five repetitions (see text for description).

Monitoring of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) changes in soybean cell suspension cultures expressing the Ca²⁺ sensing aequorin system. A, Enhancement of [Ca²⁺]_c determined upon treatment with increasing concentrations (2, 10, 50, and 200 μg mL⁻¹)of N-linolenoyl-Gln. B, Comparison of the amino acid configurations (l- or d-Gln and l- or d-Glu, respectively) of the linolenic acid conjugates (50 μg mL⁻¹) on their [Ca²⁺]_c increasing activity. C, Effect of various N-acyl Glns (50 μg mL⁻¹) on the [Ca²⁺]_c increase: N-linolenoyl-Gln, N-(15,16-epoxyoctadeca-9,12-dienoyl)-l-Gln, (15,16-epoxy-Gln), 17-hydroxylinolenoyl-l-Gln (volicitin). D, Effect of the detergent SDS (25 μg mL⁻¹) on the [Ca²⁺]_c increase in soybean cell cultures.

Time-course of the V_m variations in palisade cells distant 5, 30, and 60 mm from the bite activity of S. littoralis. The feeding larva induces a series of V_m variations leading to V_m depolarization after about 15 min from the onset of the feeding activity. In particular, when V_m was taken at an average distance of 5 mm, a strong and transient hyperpolarization occurred within 5 min after the herbivore bite, followed by a constant depolarization. The same pattern was observed when V_m was measured at a distance of 30 mm from the bite zone, but depolarization was higher than in cells at 5 mm distance. In cells 60 mm distant from the bite zone, V_m depolarization occurred within 2 to 3 min after the bite, and no hyperpolarization was observed. MW = V_m value after mechanical wounding; OL = V_m value of the opposite leaf.

False-color image analysis reconstructions from confocal laser scanning microscope observations and fluochemical intracellular Ca²⁺ determination. The green fluorescence refers to binding of Fluo-3 AM with Ca²⁺, whereas the chloroplasts are evidenced by a bright red color caused by chlorophyll fluorescence. A, Portion of a Lima bean leaf incubated with 5 mm Fluo-3 AM in 50 mm MES buffer in the presence of 0.5 mm Ca²⁺ attacked by the herbivore S. littoralis. B, Portion of a Lima bean leaf bite zone after incubation with 5 mm Fluo-3 AM in 50 mm MES buffer deprived of Ca²⁺. The green fluorescence is considerably lower than in A. C, Portion of a Lima bean leaf bite zone incubated with 5 mm Fluo-3 AM in 50 mm MES buffer in the presence of both 0.5 mm Ca²⁺ and 100 μm Verapamil. The fluorescence is decreased in the presence of the channel blocker Verapamil, with respect to A. D, Portion of a Lima bean leaf mechanically wounded after incubation with 5 mm Fluo-3 AM in 50 mm MES buffer in the presence of 0.5 mm Ca²⁺. Metric bars are indicated on pictures.