Exogenous Abscisic Acid Can Influence Photosynthetic Processes in Peas through a Decrease in Activity of H+-ATP-ase in the Plasma Membrane

Abstract

Abscisic acid (ABA) is an important hormone in plants that participates in their acclimation to the action of stressors. Treatment by exogenous ABA and its synthetic analogs are a potential way of controlling the tolerance of agricultural plants; however, the mechanisms of influence of the ABA treatment on photosynthetic processes require further investigations. The aim of our work was to investigate the participation of inactivation of the plasma membrane H⁺-ATP-ase on the influence of ABA treatment on photosynthetic processes and their regulation by electrical signals in peas. The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10^-5 M). The combination of a Dual-PAM-100 PAM fluorometer and GFS-3000 infrared gas analyzer was used for photosynthetic measurements; the patch clamp system on the basis of a SliceScope Pro 2000 microscope was used for measurements of electrical activity. It was shown that the ABA treatment stimulated the cyclic electron flow around photosystem I and decreased the photosynthetic CO₂ assimilation, the amplitude of burning-induced electrical signals (variation potentials), and the magnitude of photosynthetic responses relating to these signals; in contrast, treatment with exogenous ABA increased the heat tolerance of photosynthesis. An investigation of the influence of ABA treatment on the metabolic component of the resting potential showed that this treatment decreased the activity of the H⁺-ATP-ase in the plasma membrane. Inhibitor analysis using sodium orthovanadate demonstrated that this decrease may be a mechanism of the ABA treatment-induced changes in photosynthetic processes, their heat tolerance, and regulation by electrical signals.

Keywords: CO2 assimilation; H+-ATP-ase; abscisic acid (ABA); electrical signals; photosynthesis; photosynthetic heat tolerance; photosynthetic regulation; variation potential.

Figure 1

Schemes of different variants of experiments which were used in the current work. Fourteen to 21-day-old pea seedlings were investigated. There were four experimental variants, including an analysis of the exogenous abscisic acid (ABA) influence on (i) photosynthetic parameters under illumination by the blue actinic light (460 nm, 239 µmol m⁻²s⁻¹); (ii) photosynthetic heat tolerance; (iii) changes in photosynthetic parameters and electrical activity, induced by local burning; (iv) the metabolic component of the resting potential, which was related to the activity of H⁺-ATP-ase in the plasma membrane. In separate series of the experimental variants, the activity of H⁺-ATP-ase in the plasma membrane could be preliminarily decreased (leaves were preliminarily incubated in water solution with a moderate concentration of sodium orthovanadate ((OV), 0.5 mM, 2 h); this treatment was used for imitating the influence of exogenous ABA and for the modification of ABA-induced photosynthetic changes. Measurement of the metabolic component of the resting potential was based on fast inactivation of the H⁺-ATP-ase (minutes) under the action of the high concentration of OV (5 mM); OV was added into solution, which was placed in contact with investigated plant cells, and changes in electrical potential were measured. Local burning of the first mature leaf was induced by a flame (3–4 s, about 1 cm²) [42,43,47,50,53,55]; we did not analyze parameters of propagation of burning-induced electrical signals (variation potential, VP) in detail. Photosynthetic A_CO2 was calculated as the difference between the CO₂ assimilation rate before the termination of illumination by the actinic light (after 10 min of illumination) and this rate 5 min after the termination (after 5 min of dark conditions).

Figure 2

Quantum yields of photosystems I (Φ_PSI) (a) and II (Φ_PSII) (b), the non-photochemical quenching of chlorophyll florescence (NPQ) (c), the cyclic electron flow around photosystem I (CEF) (d), the leaf water conductance (g_H2O) (e), and the photosynthetic assimilation of CO₂ (A_CO2) (f) after the ABA treatment in pea seedlings (n = 5-15). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before photosynthetic measurements; control plants were treated with equal volumes of water. Photosynthetic parameters and leaf water conductance were measured after 10 min of illumination by blue actinic light (239 µmol m⁻²s⁻¹) in the second mature leaf. *, difference between experiment and control plants is significant (p < 0.05).

Figure 3

Influence of the ABA treatment on the photosynthetic assimilation of CO₂ (A_CO2) after heating in pea seedlings (n = 5–15). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before heating; plants without the ABA treatment were treated with equal volumes of water. Seedlings were heated from between 23 and 24 °C to 48 °C for 30 min using a thermostat. The photosynthetic CO₂ assimilation was measured after 10 min of illumination by blue actinic light (239 µmol m⁻²s⁻¹); photosynthetic measurements were performed 1 day after heating in the second mature leaf.

Figure 4

(a) Microelectrode record of burning-induced variation potential (VP) in the leaf of a control pea seedling. (b) Microelectrode record of burning-induced variation potential (VP) in the leaf of a seedling 1 day after the ABA treatment. (c) Average amplitudes of VP in control seedlings and seedlings after the ABA treatment (n = 5). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before electrical measurements; control plants were treated with equal volumes of water. Electrical measurements were performed in the second mature leaf. Variation potentials were induced (burning of the first mature leaf by a flame, 3-4 s, about 1 cm²) 1.5 h after plant fixation for measurement. A_VP was calculated as the difference between maximal and initial values of the membrane potential. *, difference between experiment and control plants is significant (p < 0.05).

Figure 5

(a) Changes in quantum yields of photosystems I (Φ_PSI) and II (Φ_PSII), the non-photochemical quenching of chlorophyll florescence (NPQ), and the photosynthetic assimilation of CO₂ (A_CO2) in the leaf of a control pea seedling after local burning. (b) Changes in quantum yields of photosystems I (Φ_PSI) and II (Φ_PSII), the non-photochemical quenching of chlorophyll florescence (NPQ), and the photosynthetic assimilation of CO₂ (A_CO2) in the leaf of an ABA-treated pea seedling after local burning. (c) Average magnitudes of these changes in photosynthetic parameters in control seedlings and seedlings after the ABA treatment (n = 5-7). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before photosynthetic measurements; control plants were treated with equal volumes of water. Photosynthetic measurements were performed in the second mature leaf; illumination by blue actinic light (239 µmol m⁻²s⁻¹) was used. Local burning of the first mature leaf by a flame (3-4 s, about 1 cm²) was performed 1.5 h after plant fixation for measurement. *, difference between experiment and control plants is significant (p < 0.05).

Figure 6

A scatter plot showing the rates of photosynthetic CO₂ assimilation (A_CO2) and magnitudes of local burning-induced decreases in A_CO2 in pea seedlings (n = 12). R² and R are determination and correlation coefficients, respectively.

Figure 7

Relative values of the metabolic component of the resting potential in pea seedlings after treatment by ABA and a moderate concentration of sodium orthovanadate (OV) (n = 5–7). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before electrical measurements; control plants were treated with equal volumes of water. The preliminary treatment with the moderate OV concentration in the second mature leaf in seedlings was performed by incubation of the leaf (2 h) in a solution of OV (0.5 mM); after that, this leaf was dried by filter paper and was used for the measurement of electrical activity. Similar treatment by water was used in the control. Measurements of the metabolic component of the resting electrical potential across the plasma membrane were performed with the addition of a high concentration of OV (5 mM) during the electrical record; only short-term changes in the membrane potential were analyzed (see Figure S2 for details). The metabolic component was formed by the active transport of H⁺ across the plasma membrane, i.e., it was strongly related to H⁺-ATP-ase activity in the plasma membrane. Relative values of the metabolic components were calculated as the ratio of experimental values to control ones. *, difference between experiment and control plants is significant (p < 0.05).

Figure 8

Influence of the modification of the H⁺-ATP-ase activity by sodium orthovanadate (OV) on photosynthetic CO₂ assimilation after the ABA treatment (n = 5–10). The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before photosynthetic measurements; control plants were treated with equal volumes of water. The preliminary OV treatment of the second mature leaf in seedlings was performed by incubation of the leaf (2 h) in a solution of OV with a moderate concentration (0.5 mM); after that, this leaf was dried by filter paper and used for photosynthetic measurements. Similar treatment by water was used in the control. Photosynthetic parameters were measured after 10 min of illumination by blue actinic light (239 µmol m⁻²s⁻¹) in the second mature leaf.

Figure 9

Influence of the modification of H⁺-ATP-ase activity by sodium orthovanadate (OV) on the photosynthetic heating tolerance of pea seedlings in the control group and after the ABA treatment (n = 5-14). (a) Rates of the photosynthetic CO₂ assimilation (A_CO2) in different variants of the experiment. (b) Heating-induced decreases in A_CO2 in control and OV treatment groups. The ABA treatment of seedlings was performed by spraying them with aqueous solutions (10⁻⁵ M) 1 day before heating; control plants were treated with equal volumes of water. The preliminary OV treatment of the leaves of seedlings was performed by incubation of the leaf (2 h) in a solution of OV with a moderate concentration (0.5 mM); after that, this leaf was dried by filter paper and seedlings were heated. Similar treatment by water was used in the control. Seedlings were heated from 23–24 °C to 48 °C for 30 min using a thermostat. The photosynthetic CO₂ assimilation was measured after 10 min of illumination by blue actinic light (239 µmol m⁻²s⁻¹); photosynthetic measurements were performed 1 day after heating in the second mature leaf.

Figure 10

Influence of the modification of H⁺-ATP-ase activity by sodium orthovanadate (OV) on the local burning-induced decreases in CO₂ assimilation. (a) Local burning-induced changes in A_CO2 in the leaf of a control seedling. (b) Local burning-induced changes in A_CO2 in the leaf of a seedling after vanadate treatment. (c) Average magnitudes of local burning-induced A_CO2 decreases (n = 5–6). The preliminary OV treatment of the leaves of seedlings was performed by incubation of the leaf (2 h) in a solution of OV with a moderate concentration (0.5 mM); after that, this leaf was dried by filter paper and photosynthetic measurements were performed. Similar treatment by water was used in the control. Photosynthetic measurements were performed in the second mature leaf; illumination by blue actinic light (239 µmol m⁻²s⁻¹) was used. Local burning of the first mature leaf by a flame (3–4 s, about 1 cm²) was performed 1.5 h after plant fixation for measurement. *, difference between experiment and control plants is significant (p < 0.05).

Figure 11

A hypothetical scheme of the potential modes of participation of the H⁺-ATP-ase of the plasma membrane in the influence of the spraying of plants with exogenous ABA on the photosynthetic CO₂ assimilation, heat tolerance of photosynthetic processes, and their regulation by electrical signals (see text for a detailed description). Colored boxes mark the results which are shown in the current work.