Selenium preserves cytosolic-Ca2+ homeostasis in olive callus cells during oxidative stress

Selenium (Se) is not essential for plant nutrition, however, growing evidence suggests a role of this element against oxidative stress. Here, we investigated the antioxidant effect of Se in an experimental system consisting of suspensions of olive callus cells exposed to hydrogen peroxide. Due to the disruptive capacity of oxidative stress on cytosolic Ca2+ homeostasis, Se treatments were simply and effectively assessed with cytosolic Ca2+ measurements. Se did not alter cell morphology, nor interfered with fluorometric determinations, suggesting a suitable model for monitoring alterations of cytosolic Ca2+. Furthermore, we used thapsigargin (TG), a known inhibitor of the Ca2+-ATPases of the endoplasmic reticulum, to establish whether Se had a specific impact on oxidative stress. We found that Se antagonized H2O2-mediated perturbations of cytosolic Ca2+, but was unable to offset TG-mediated disruptions. We conclude that Se merits consideration in strategies designed to tackle abiotic stress leading to oxidative insults. Se preserves cytosolic-Ca2+ levels from the disruptive effect of oxidative stress induced by hydrogen peroxide in olive callus cells.


Introduction
The implication of selenium (Se) in human health is wellknown. Whether naturally present in foods, taken with supplements, or biofortified in crops, Se has been used for years to prevent chronic diseases (Boosalis 2008;Rayman 2008) besides toxicity risks that, conversely, may arise from highintake regimens (Rees et al. 2013).
Although not generally considered essential to plants, growing evidence points toward a major antioxidative role of Se in abiotic stress raised by an excessive occurrence of reactive oxygen species (ROS) (Lyons et al. 2009;Prins et al. 2011;Tedeschini et al. 2015). In plants, physiological levels of ROS are relevant to a large array of biochemical reactions and cell signaling pathways implicated in major biological processes, such as photosynthesis, development, growth, programmed cell death, and defense mechanisms (Hancock et al. 2001;Laloi et al. 2004;Kwak et al. 2006). On the other hand, abnormal ROS accumulation can cause damages to cell structures, as well as metabolic imbalances including cytosolic-Ca 2+ homeostasis.
The connection of Ca 2+ to plant biology is very diverse, given a structural involvement in cell walls and membranes, and the physiological significance of cytosolic-Ca 2+ in the role of second messenger (Hetherington and Brownlee 2004;Clapham 2007;Dodd et al. 2010;Steinhorst and Kudla 2013;Del Pino et al. 2019). Cytosolic-Ca 2+ enters plant cells through several channels embedded in the plasma membrane to promote vertical growth, fertilization, and pollen tube formation, and to challenge abiotic stresses (Lazzaro et al. 2005;Campanoni and Blatt 2007;Cheung and Wu 2008;Michard et al. 2009). Cytosolic-Ca 2+ homeostasis is finely controlled, and maintained within nanomolar concentrations to modulate cell signaling-dependent events; however, disruptions caused by excessive ROS levels may therefore lead to pathological dysfunctions, shown for example by abnormal pollen germination (Yan et al. 2006;Brini et al. 2013;Görlach et al. 2015;Orrenius et al. 2015;Del Pino et al. 2019). Recently, we found that Se maintains physiological cytosolic-Ca 2+ levels in pollen of olive trees subject to oxidative stress, and that pollen from Se-treated trees, unlike the untreated counterpart, retains germination rates for at least 20 months (Del Pino et al. 2019;Pino et al. 2021). Taken together, these observations recapitulate the functional implication of cytosolic-Ca 2+ homeostasis, and suggest a beneficial impact of Se on olive trees during oxidative stress (Steinhorst and Kudla 2013;Görlach et al. 2015).
The olive tree is the most widespread fruit crop in the Mediterranean region, and its improvement in productivity by conventional and biotechnological approaches and in the treatment of abiotic stresses is currently in demand. In this context, several authors have highlighted the possibility of applying in vitro techniques such as micropropagation (Rugini et al. 1984;Zacchini et al. 2004), somatic embryogenesis (Rugini et al. 1995;Trabelsi et al. 2011) to the olive tree, callus and suspension cell cultures (Shibli et al. 2002;Gentile et al. 2014, Iori et al. 2015. Here, we used suspended olive callus cells (OCCS) to test the effects of oxidative stress on cytosolic Ca 2+ metabolism and the ability of selenium to antagonize hydrogen peroxide.
Additionally, our results support the notion that OCCS represent an experimental resource to monitor over time redox oscillations and, more generally, the efficacy of antioxidants against redox-related insults.

Preparation of olive callus cells suspension (OCCS)
Approximately 1 g of calli was placed in a Petri dish, minced with a scalpel, and then treated with 6 mL of trypsin for approximately 10 min. The supernatant was collected in a Falcon and then centrifuged at 3000 rpm×4 min to remove trypsin, after which the pellet was washed in 6 mL PBS. After another centrifugation (3000 rpm×4 min), the pellet was ground in a Potter-Elvehjem tissue homogenizer, resuspended in PBS, and then centrifuged (3000 rpm, 4 min). Lastly, the pelleted material was resuspended in PBS and manually counted in a hemocytometer to prepare 10 6 OCCS aliquots subsequently used for Cytosolic-Ca 2+ measurements.

Data analysis
Evaluations were performed using the GraphPad Prism 6.03 software (La Jolla, CA, USA). Assumptions of variance were assessed using the Levene's test (homogeneity of variance) and the D'Agostino-Pearson omnibus normality test (normal distribution). Results are expressed as mean values ± standard error of the mean (SEM). The significance of differences was analysed by the Fisher's least significant differences test following analysis of the variance according to the 2-way split-plot design with complete randomisation (Figs. 2 and 3) and 3-way split-plot with complete randomisation (Figs. 4 and 5). Statistical significance implied p-values less than 0.05.

Histological analysis
Consistent with a previous report (Chiavegatto et al. 2015), calli displayed (a) vacuolated cells with numerous intercellular spaces; (b) vacuolated cells with transverse septa indicative of mitotic activity; and (c) crushed cells morphologically similar to cambium cells ( Fig. 2A).
On the other hand, semithin sections of OSSC showed one cell type only, namely, small cells with

Oxidative stress and disruption of cytosolic-Ca 2+ homeostasis in olive callus cells suspension (OCCS)
Oxidative stress in OCCS was induced by hydrogen peroxide ranging between 5mM and 40 mM. Without Ca 2+ in the incubation medium (HBSS Ca 2+ free), hydrogen peroxide caused a dose-response increase of cytosolic-Ca 2+ levels (Δ[Ca 2+ ] c , nM) with maximal effects at 10 mM and 20 mM concentrations (Fig. 3). When 1mM CaCl 2 was added to the incubation medium, the amount of Ca 2+ entry from the extracellular space was significantly reduced by H 2 O 2 with maximal effects in the concentration range of 10 to 40 mM (Fig. 3).

Effectof selenium on cytosolic-Ca 2+ homeostasis in OCCS subject to oxidative stress
Na-selenate (1.0-13.6 µM) had a negligible effect on cytosolic Ca 2+ levels in OCCS (data not shown), however, under Ca 2+ free conditions (0 mM), Na-selenate ranging between 3.4 µM and 13.6 µM neutralized most of the effects caused by hydrogen peroxide on cytosolic-Ca 2+ (Fig. 4). On the other hand, in OCCS cultured in medium containing 1mM CaCl 2 , presence of selenium significantly attenuated the peroxide-mediated decrease in cytosolic-Ca 2+ , accounting to 56% and 46% in the absence and presence of 3.4mM-13.6mM Na-selenate, respectively (Fig. 4).

Effects of thapsigargin (TG) on Ca 2+ homeostasis of olive callus cells suspension (OCCS) under oxidative stress
In the absence of H2O2, thapsigargin (TG) increased cytosolic-Ca 2+ in OCCS with a maximal effect at 20 µM (Fig. 5A) and did not elicit Ca 2+ entry from the extracellular medium when cells were exposed to 1mM CaCl 2 (Fig. 5A).
Under oxidative stress, the increase in [Ca 2+ ] c produced by hydrogen peroxide was reduced by TG in a dosedependent manner under Ca 2+ free conditions (Fig. 5B). Conversely, TG increased Ca 2+ entry by ~ 30% in OCCS exposed to 1mM CaCl 2 (Fig. 5B).

Effects of selenium and/or thapsigargin (TG) on Ca 2+ homeostasis of olive callus cells suspension (OCCS) in oxidative stress
Na-selenate (3.4 µM) had negligible effects on cytosolic Ca 2+ under basal conditions and only partially interfered with the effects of thapsigargin on Ca 2+ homeostasis, under Ca 2+ free conditions (Fig. 6A).
Selenium does not perturb Ca 2+ entry in the presence of TG and shows no effect on the entry of Ca 2+ from the extracellular medium when cells were exposed to 1mM CaCl 2 (Fig. 6A).
The reduction of Ca 2+ entry produced by H 2 O 2 was partially antagonized by Na-selenate in the presence of TG (Fig. 6B). ) were used to treat cells before (0 mM CaCl 2 ) and after the addition of Ca 2+ (1mM CaCl 2 ) in the incubation medium. Data are expressed as means ± SEM from 4 independent tests. Different letters indicate statistically significant differences (p < 0.05)

Discussion
Abiotic stresses hurt agriculture by impacting crop quality and overall productivity (Change 2012). A stress can pose a threat on growth, germination rates, and pollen viability (Hasanuzzaman et al. 2013), and cause premature leaf senescence upon alterations of the respiratory and photosynthetic systems and abnormalities in membrane permeability. A typical consequence of abiotic stress is the accumulation of ROS (Paupière et al. 2014). Concentrations of ROS that exceed the capacity of cell scavenging mechanisms, lead to irreparable damage across a large array of cellular components (Hancock et al. 2001). Notably, the reactive nature of ROS deregulates Ca 2+ signaling, which plays a vital role in processes such as growth and fertility (Lazzaro et al. 2005;Michard et al. 2009;Zinn et al. 2010).
Selenium is a plant micronutrient with antioxidant properties that has an impact on the connection between ROS and Ca 2+ signaling (Lyons et al. 2009;Prins et al. 2011;Tedeschini et al. 2015).
In this context, several authors have highlighted the possibility of applying in vitro techniques to the olive tree such as micropropagation (Rugini et al. 1984;Zacchini et al. 2004), somatic embryogenesis (Rugini et al. 1995;Trabelsi et al. 2011), callus and suspension cell cultures (Shibli et al., 2002;Gentile et al. 2014). Particular attention was paid to the callus culture technique to characterize stress-tolerant cultivars (Shibli et al. 2002) and to study the production of bioactive compounds (Saimaru et al. 2007;Gentile et al. 2014).
In this work, the effect of selenium on Ca 2+ homeostasis disrupted by oxidative stress was tested for the first time in olive callus cells suspension (OCCS) obtained with a procedure ensuring maintenance of cell morphology and viability, shown by both optical microscope images and the extent of the fluorescence signal. Given that a cell suspension is a key requirement for cytosolic Ca 2+ determinations by FURA staining, we demonstrated that OCCS can therefore be used as a reproducible biological source for monitoring fluctuations of cytosolic Ca 2+ during oxidative stress.
Our results confirmed that oxidative stress, induced in OCCS by hydrogen peroxide, determined an increase of cytosolic-Ca 2+ and a reduction of Ca 2+ entry from the extracellular medium, thereby altering Ca 2+ homeostasis. However, pre-treatment of OCCS with Na-selenate prevented (1 mM CaCl 2 ) in the incubation medium. Data are expressed as means ± SEM from 4 independent tests. Different letters indicate statistically significant differences (p < 0.05) Fig. 7 UV-VIS thapsigargin, Na-selenate, thapsigargin +Na-selenate spectra the disruptive effects of hydrogen peroxide on (Ca 2+ ) c , and restored, at least in part, the entry of Ca 2+ from the medium.
To gain insights on the mechanisms associated with the toxic effects of hydrogen peroxide, OCCS were treated with thaspigargin (TG), an inhibitor of cytosolic-Ca 2+ sequestration (Thi Quynh Doan and Brogger Christensen 2015). TG is an apoptotic agent, not involved in oxidative stress, which inhibits endoplasmic reticulum (ER) Ca 2+ -ATPases and, thus, perturbs cytosolic Ca 2+ homeostasis (Thi Quynh Doan and Brogger Christensen 2015). As expected, TG elevated cytosolic Ca 2+ levels and depleted Ca 2+ stored in the ER. Since a largely reduced presence of Ca 2+ in cellular stores shows that hydrogen peroxide failed to disrupt cytosolic-Ca 2+ homeostasis, our data suggest that H 2 O 2 deregulates Ca 2+ exit from the ER.
Selenium antagonizes the effects of hydrogen peroxide on Ca 2+ homeostasis. The same was not observed upon exposure to TG. Indeed, in the presence of selenium, TG showed less activity on cytosolic Ca 2+ . Comparison of the UV-VIS spectra of thapsigargin with thapsigargin +Na-selenate showed differences in absorbance. This suggests that TG and Na-selenate enter the SERCA site as a complex. X-ray structure of SERCA showed that hydrophobic interactions and water molecules are present with an important role for the correct localization of TG at the binding site of SERCA (Toyoshima and Nomura 2002).
This suggests that the TG+Na selenate complex, influencing the formation of the stabilizing SERCA hydrogen bonds, causes a lower affinity for Ca 2+ .
Since cytosolic Ca 2+ homeostasis also depends on extracellular ion entry (Ca 2+ entry), it appears that hydrogen peroxide deregulates this process as well. Thus, one may conclude that selenium not only elicits a release of Ca 2+ from the ER, but also prompts Ca 2+ entry in the presence of H 2 O 2 .
In conclusion, selenium contributes to the maintenance of Ca 2+ homeostasis under oxidative stress in OCCS, but does not exhibit the same effect when Ca 2+ homeostasis is perturbed by thapsigargin. Since selenium is not toxic under the concentrations applied, it may be considered a valid candidate in the prevention, or treatment, of oxidative stress.
Funding Open access funding provided by Università degli Studi di Perugia within the CRUI-CARE Agreement. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no relevant financial or nonfinancial interests to disclose.

Ethical statement Not applicable.
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