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Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.5 JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.5 Here, we report that a plasma membrane potential (Vm) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter Vm but the perfusion of JA and the polyamines significantly increased Cad and Put Vm depolarization. When JA was perfused with polyamines, a negative correlation was found between Vm depolarization and the number of amino group of the polyamines tested.
Polyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.1–4 We recently reported that exogenously applied polyamines ~diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)] induce volatile emission in Lima bean leaves.5 Membrane potentials (Vm) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.
The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in Vm.6 The plasma membrane potential (Vm), which lies in the range of −50 to −200 mV in Lima bean leaves,7 may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.
Measurement of Vm were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).7 Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different Vm depolarization, depending on the polyamine. In general, a Vm depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd Vm depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad Vm depolarization (p = 0.435). In all cases, Vm depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the Vm was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of Vm depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min−1; R = 0.96), equal for Put and Cad (4.8 mV min−1; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min−1; R = 0.96).
Perfusion with JA caused a slight and not significant (p = 0.332) Vm depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in Vm depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of Vm depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min−1; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min−1, R = 0.99; Spd: 13.49 mV min−1, R = 0.99) and lower for Spm (1.34 mV min−1; R = 0.93). For JA the rate of Vm depolarization was 0.19 mV min−1 (R = 0.96). With the addition of JA, a negative correlation was found between Vm depolarization and the number of amino group of the polyamines tested.
Since ion fluxes through channels directly influence Vm, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.8 In the present study, rapid and reversible Vm depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the Vm may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.9 Liu et al.10 showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.1,11 Both mechanisms can be correlated to the observed Vm depolarization, and the positive correlation between intracellular Ca2+ concentration5 and Vm depolarizing activity of polyamines confirms the involvement of Ca2+ during polyamine uptake.11
This work was supported by: Grant-in-Aid for Scientific Research (S) (No. 19101009 to J.T.); Global COE Program A06 of Kyoto University; CEBIOVEM (Centre of Excellence for Plant and Microbial Biosensing of the University of Turin to M.E.M.).
Previously published online: www.landesbioscience.com/journals/psb/article/10848