Electric signaling and pin2 gene expression on different abiotic stimuli depend on a distinct threshold level of endogenous abscisic acid in several abscisic acid-deficient tomato mutants

Abstract

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.

Figure 1

Mechanical wounding, electrical current, and heat treatments initiate the local and systemic accumulation of Pin2 mRNA. Wild-type (MM) and ABA-deficient (notabilis, flacca, and sitiens) mutant tomato plants were wounded or treated with electrical current or heat. Six hours after treatment, total RNA was isolated from the treated leaves (L) and from leaves located distal (S) to the treated ones. The autoradiogram shows the result of a gel-blot hybridization of total RNA (10 μg per slot) against radioactive Pin2 (Sanchez-Serrano et al., 1986).

Figure 2

ABA content in tomato plants. Wild-type (MM) and ABA- deficient (notabilis, flacca, and sitiens) mutant tomato plants were wounded or treated with electrical current or heat. Both directly treated (local) and nontreated distal leaves (systemic) were harvested after 6 h. Endogenous levels of ABA were determined as described in Methods and are indicated as means ± se (n = 5). FW, Fresh weight.

Figure 3

Effect of wounding, electrical current, and heat treatment on endogenous levels of JA. Wild-type (MM) and ABA-deficient (notabilis, flacca, and sitiens) mutant tomato plants were wounded or treated with electrical current or heat. Both directly treated (local) and nontreated distal leaves (systemic) were harvested after 6 h. Endogenous levels of JA were determined as described in Methods and are indicated as means ± se (n = 5). FW, Fresh weight.

Figure 4

Characteristic membrane potential relaxation kinetics in the main vein of a leaf of ABA-deficient tomato mutants caused by mechanical wounding (indicated by an arrow). Membrane potential recordings caused by current application and heat treatment resembled those after mechanical wounding (data not shown). The results shown are characteristic examples of a pool of 30 measurements per plant.

Figure 5

Characteristic membrane potential relaxation kinetics in the main vein of a wild-type tomato plant caused by exogenous application of ABA (indicated by an arrow) . The results shown are characteristic examples of a pool of 30 measurements per plant.