**1. Introduction**

A wide range of signal perception and transduction systems in plant cells is responsible for distinguishing and triggering a correct adaptive response [1,2]. Signal transduction pathways that are specific for different stressors include several steps that are shown to be rather universal. One of them is a cytosolic Ca2<sup>+</sup> elevation at internal and external signals application [3–5]. Plant cells carefully maintain a low Ca2<sup>+</sup> concentration in the cytosol and a significant gradient between the cell wall and a number of organelles. This balance is actively regulated by a variety of membrane transport systems (recently reviewed in [6]). Another well-documented primary signaling event is the accumulation of reactive oxygen species (ROS) [7–10]. The appearance of ROS caused by different stressors triggers a wide spectrum of reactions and is quickly eliminated by the antioxidant machinery. Both the second messengers (Ca2<sup>+</sup> and ROS) are closely linked to each other and are involved in the transduction of different environmental stressors and internal signals such as phytohormones, regulatory proteins, RNAs and metabolites [6,7,11]. One important mechanism is the Ca2<sup>+</sup>-induced activation of NADPH-oxidase activity via its integration in sterol-rich lipid rafts [12]. On the other

hand, ROS activates Ca2<sup>+</sup> channels in the plasma membrane [3] and Ca2+-ATPases, which interfere with Ca2<sup>+</sup> homeostasis [13]. Initially, weak signals of both Ca2<sup>+</sup> and ROS were hypothesized to be amplified through the so-called ROS-Ca2<sup>+</sup> hub [6]. The change of Ca2<sup>+</sup> and ROS to an inactive state is also a necessity and supposed to be highly regulated. The cytosol Ca2<sup>+</sup> elevation triggered by a variety of internal and external signals coincides with cytosolic acidification (reviewed in [14,15]). It was questionable for some time if protons had a signaling role, probably because of its involvement in metabolism. It is well known that Ca2<sup>+</sup> elevation in the cytosol affects the pH level. The duration and intensity of Ca2<sup>+</sup> increases might vary and thus determine the specificity of response to diverse signals (stressors, hormones, light, etc.). Different systems of H<sup>+</sup> transport through the plasma membrane and tonoplast are involved. Proton pumps are supposed to be regulated on transcriptional and posttranslational levels, which make the H<sup>+</sup> signature highly specific.

Electrolyte leakage is another process accompanying transient Ca2+, ROS and proton increases under stress conditions. A number of experimental data reveals that electrolyte leakage is mainly defined as K<sup>+</sup> efflux from plant cells [16,17].

Potassium is essential for plant cells/organisms in many aspects. It is a well-known macro-nutrient. Deficiency of K<sup>+</sup> results in growth arrest especially in seedlings and young organs. This ion is important for plant metabolism due to its facility to activate more than 70 enzymes [17,18]. Besides that, K<sup>+</sup> serves as a charge-balancing ion, which plays an important role in the transport through the plasma membrane under the limitation of ATP and the depolarization of the membrane potential. The K<sup>+</sup> gradient maintains turgor and serves as a source of energy to stimulate sucrose loading into the phloem [19,20]. Accumulated evidence indicates that the efflux depends on the type and the intensity of the stressor as well as on affected plant species and tissue. The cytosolic K<sup>+</sup> concentration in plant cells is about 70–200 mM [21]. Several transporters have been shown to be involved in K<sup>+</sup> accumulation: the High Affinity K<sup>+</sup> transporter (HAK)–K<sup>+</sup> uniporter and the Arabidopsis K<sup>+</sup> Transport system 1 (AKT1)–K+/H<sup>+</sup> symporter [18,22]. These processes require energy and depend on the external K<sup>+</sup> concentration and the K<sup>+</sup> vacuolar pool. The priority role in stress-induced K<sup>+</sup> leakage is given to another system: Gated Outward Rectifying K<sup>+</sup> efflux (GORK) channels [23]. By activation, it decreases the cytosolic potassium concentration to 10–30 mM [21]. The activity of these channels is sensitive to membrane potential depolarization through a clustering mechanism [24]. A number of experimental data combined with a bioinformatics approach suggest a possible ligand regulation of K<sup>+</sup> flux through GORK channels [25]. Cyclic nucleotides (CNs), gamma-aminobutyric acid (GABA), G proteins, protein phosphatases, inositol, ROS and ATP are on a list of potential GORK regulating ligands. The presented data even stronger introduced both K<sup>+</sup> and GORK in signaling cascades triggered by stress factors and led to the conclusion that potassium fulfills the role of a second messenger [17,25]. A signaling role of potassium is well documented for salt stress [18].

Oxygen deficiency is another stress factor that affects K<sup>+</sup> efflux and causes severe damage to plant organisms [26–30]. Surprisingly, plants known as strict aerobic organisms might be affected not only by an external lack of oxygen. Different tissues or even groups of cells experience hypoxia conditions during normal plant development [31]. Thus, it becomes even more important to discover the steps of early hypoxia signal transduction. The mechanism of oxygen sensing in plants and animals are strictly diverse. However, low oxygen regulates the function of various K<sup>+</sup> channels in mammals. Recently, plant cell ion channels have been identified as potential candidates for low oxygen sensing in flooded roots [32]. However, additional studies are required to estimate a possible modulation of K<sup>+</sup> transport through an ERF-VII-mediated response to a lack of oxygen. Nevertheless, hypoxic/anoxic environment causes K<sup>+</sup> efflux from plant cells and GORK channels are supposed to play a crucial role in this process [25]. Under hypoxic stress, cytosolic K<sup>+</sup> participates in the regulation of several physiological processes possibly including the formation of aerenchyma [33].

Oxygen deprivation triggers Ca2<sup>+</sup> signaling. The alteration in [Ca2<sup>+</sup>]cyt is a fast and intensive reaction for limitation in oxygen supply and energy deficiency [34–37]. Ca2<sup>+</sup> elevation in cytosol coincides with acidification [15]. A probable reason is the hypoxia-induced inhibition of the plasma membrane H+-ATPase and tonoplast H+-ATPase activities due to the lack of ATP.

Signaling via K<sup>+</sup> efflux is transient and therefore the timing of this event should be evaluated. It was found that plant species and even plant organs differ in their tolerance to hypoxia. Some data revealed that the intensity of K<sup>+</sup> efflux corresponded to plant sensitivity to oxygen deprivation and depended on metabolic activity [30]. It would be of interest to integrate [K<sup>+</sup>]cyt into the schedule of other intracellular signaling events such as Ca2<sup>+</sup> elevation and acidification under a lack of oxygen. Earlier we provided a comparison of calcium signaling during anoxia in two well-known agricultural plants such as rice and wheat, which differ in tolerance to oxygen limitation [35]. Rice cells were shown to be more reactive to oxygen depletion and depended on both external and internal Ca2<sup>+</sup> stores. This study focuses on a possible alteration of intracellular potassium concentration, [K<sup>+</sup>]cyt, and cytosolic pH, pHcyt, during anoxic signal transduction in the protoplasts from wheat and rice.

The mentioned ions have not only a signaling role but also are very important in the regulation of metabolic processes including those at stress conditions. We therefore also estimated the cell-level events at a whole plant level. We investigated the ion changes after reoxygenation as well. It is of special interest how proton and Ca2<sup>+</sup> accumulation inside cells and active potassium efflux would reflect processes during long-time stress applications and regulate the ion exchange with an external medium.

#### **2. Results**
