Calcium-dependent changes in the activity of antioxidant enzymes and heat resistance of wheat seedlings under the influence of exogenous putrescine

Polyamines are important stress plant metabolites involved in the regulation of redox homeostasis and other signaling processes. Polyamines can affect redox homeostasis by binding free radicals and participating in the regulation of gene expression of antioxidant enzymes. However, their metabolism produces hydrogen peroxide and other reactive oxygen species (ROS). A separate component of the physiological activity of polyamines may be their effect on the state of ion channels. It is known that there are complex bonds between ROS and calcium ions as signal mediators. However, the functional interaction between ROS and calcium ions in the implementation of the effects of polyamines on plant cells has been studied very poorly. Moreover, the question of the role of such interactions in the realization of stress-protective effects of polyamines remains open. The aim of this research was to study the inhibitory methods of the involvement of different pools of calcium in the regulation of the formation of hydrogen peroxide, the activity of antioxidant enzymes and inducing putrescine thermostability of wheat seedlings. The object of the study was etiolated seedlings of soft winter wheat (Triticum aestivum L.) of Doskonala variety. Three-day-old seedlings were treated with putrescine at a concentration of 1 mM. In certain experimental variants, the seedlings were treated with calcium antagonists - EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid - extracellular Ca²⁺ chelator) and neomycin - an inhibitor of phospholipase C-dependent calcium intake into the cytosol from intracellular compartments, as well as with the indicated inhibitors in combination with putrescine. In the roots of seedlings some time after treatment with test compounds, we determined hydrogen peroxide content and the activity of antioxidant enzymes. One day after seedling treatment with putrescine, calcium antagonists and a combination of effectors, the seedlings were subjected to damaging heating in a water thermostat (10 min at 45 °C). 5 hours after heating, we assessed damage to the root cell membranes by the yield of compounds absorbing in the UV-B, as well as the activity of antioxidant enzymes. 2 hours after the start of root treatment with putrescine, we noted a significant increase in the content of hydrogen peroxide in them (See Fig. 1). This effect was eliminated by treatment with EGTA, but not with neomycin. The treatment of seedlings with putrescine caused a change in the activity of all three studied antioxidant enzymes in roots (superoxide dismutase - SOD, catalase, and guaiacol peroxidase) (See Table). The increased activity of these enzymes in the roots of seedlings treated with putrescine was also observed after damaging heating. The calcium ion chelator completely eliminated the putrescine-induced effect of increasing the activity of all three studied enzymes (See Fig. 2). Neomycin almost did not affect the manifestation of the effect of increasing the activity of SOD, however, it neutralized the increase in the activity of catalase and guaiacol peroxidase caused by putrescine. The effect of heat stress on the seedlings caused an increase in the yield of substances absorbing in the UV from the root cells. Treatment with putrescine helped preserve the integrity of biomembranes. Calcium antagonists EGTA and neomycin leveled off the effect of reducing the yield of substances absorbing in the UV-B caused by putrescine (See Fig. 3). They also almost completely eliminated the putrescine-induced increase in survival of seedlings after stress. We can conclude that the induction of heat resistance of wheat seedlings by putrescine depends on the functional interaction between calcium ions and ROS as signaling agents. The activation process of the enzymatic antioxidant system, necessary for the formation of heat resistance of plants, depends on the formation of ROS, which occurs with the participation of diamine oxidase and NADPH oxidase. At the same time, the accumulation of the signal pool of hydrogen peroxide is dependent on the influx of calcium into cytosol from extracellular space. The further activation process of the antioxidant enzyme complex under the influence of putrescine treatment also depends on the influx of calcium into cytosol, not only from extracellular space, but also from intracellular compartments. The paper contains 3 Figures, 1 Table and 34 References. The Authors declare no conflict of interest.

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