The relationship between photosynthesis and a mastoparan-induced hypersensitive response in isolated mesophyll cells

Abstract

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

Figure 1

Sample of PAM chlorophyll a fluorescence yield from a suspension of isolated asparagus mesophyll cells. AL, Actinic light; MB, measuring beam; SF, saturating flash.

Figure 2

Cell viability as a function of MP concentration. Two-milliliter cell suspensions containing 12 × 10⁶ cells were incubated with either 0, 0.25, 2.5, or 25 μm MP. After 16 min of incubation with MP, the cells were incubated with Evan's blue dye for assessment of viability. Percentage cell death represents the mean of three experiments. se bars are shown.

Figure 3

Individual cell viability assayed with Evan's blue dye and fluorescein diacetate. Two 2.5-mL cell suspensions, each containing 10 × 10⁶ cells, were incubated for 16 min with and without 25 μm MP, respectively. Cells were then incubated with fluorescein diacetate in conjunction with Evan's blue dye. Photographs were taken of the same fields of view under both bright and dark fields. These photographs represent control cells under bright (A) and dark (B) fields, and MP-treated cells under bright (C) and dark (D) fields.

Figure 4

The effect of MP and Mas17 on the pH of the incubating medium. The pH of suspensions containing 4.5 × 10⁶ cells in 3 mL of 1 mm CaSO₄ was adjusted to approximately 5.0. A, The addition of 13.2 μm MP and two aliquots of 0.10 n HCl (for back-titration) are indicated by arrows. B, The addition of 13.2 μm Mas17 is indicated by an arrow. This figure is a representative of five runs.

Figure 5

The effect of MP concentration on O₂ consumption in the light. This figure shows O₂ consumption measurements compiled from five separate PAM experiments. In each experiment 2 mL of 5 mm Mes and 1 mm CaSO₄, pH 6.0, contained 3 × 10⁶ cells. Aliquots of MP were added to give the concentrations indicated.

Figure 6

The effect of Mas7 on O₂^⋅− production. Three million cells were incubated in 330 mL of 1 mm CaSO₄, pH 5.5, for 5 min. Arrow 1 indicates the addition of 5 μm Mas7 to the cell suspension. Arrow 2 indicates transfer of 200 μL of the cell suspension to the luminometer cuvette. Arrow 3 indicates an artifact due to stray light detected by the photomultiplier as the cuvette is inserted into the luminometer. This figure is a representative of six runs.

Figure 7

The effect of Mas7 on chlorophyll a fluorescence yield and O₂ level in the light. Two milliliters of 5 mm Mes and 1 mm CaSO₄, pH 6.0, contained 3 × 10⁶ cells. Far-red light (FR), actinic light (AL), 22 μm MP, and O₂ were added (↑) or removed (↓) as indicated. The inset represents relative fluorescence immediately before and after Mas7 addition. This figure is a representative of 20 runs.

Figure 8

The effect of Mas7 on q_P and q_N, respectively over time. Values of q_P and q_N shown were calculated from one representative trial. Actinic light (AL) and 22 μm Mas7 were administered as indicated by the arrows.

Figure 9

The effect of Mas7 on chlorophyll a fluorescence yield and O₂ level in the dark. Two milliliters of 5 mm Mes and 1 mm CaSO₄, pH 6.0, contained 3 × 10⁶ cells. Cell suspensions were not illuminated with actinic light. Far-red light (FR), 22 μm MP, and O₂ were added (↑) or removed (↓) as indicated. This figure is a representative of 10 runs.