Selenium-Enriched Pollen Grains of Olea europaea L.: Ca2+ Signaling and Germination Under Oxidative Stress

Abstract

Selenium (Se) shows antioxidant properties that can be exploited in plants to combat abiotic stresses caused by reactive oxygen species produced in excess (ROS). Here, we show that the Se-fertilization of olive trees with sodium selenate effectively protects the pollen from oxidative stress. Pollen isolated from plants treated with Se or from untreated controls was incubated in vitro with H₂O₂ to produce an oxidative challenge. Given the impact of ROS on Ca²⁺ homeostasis and Ca²⁺-dependent signaling, cytosolic Ca²⁺ was measured to monitor cellular perturbations. We found that H₂O₂ interrupted Ca²⁺ homeostasis only in untreated pollen, while in samples treated in vitro with sodium selenate or selenium methionine, Ca²⁺ homeostasis was preserved. Furthermore, germination rates were considerably better maintained in Se-fertilized pollen compared to non-fertilized pollen (30% vs. 15%, respectively) after exposure to 1 mM H₂O₂. The same was observed with pollen treated in vitro with Se-methionine, which is the organic form of Se, in which part of the fertigated sodium selenate is converted in the plant. Combined, our results show a close correlation between ROS, Ca²⁺ homeostasis, and pollen fertility and provide clear evidence that Se-fertilization is a potential approach to preserve or improve agricultural productivity.

Keywords: cytosolic Ca2+; olive; pollen germination; se-fertilization; se-methionine; selenium.

Figure 1

Effect of H₂O₂ (0.1–5.0 mM) on [Ca²⁺]_cp in control pollen grains in the presence (○) and absence (•) of CaCl₂ in the incubation medium. Results are expressed as Δ[Ca2+]cp (nM) to reflect signal changes detected between the start and the end of the fluorometric measurements and represent means ± SEM from four independent tests. Statistically significant differences between ○ and • are indicated by different letters, whereas identical letters highlight non-significant trends.

Figure 2

Effects of sodium selenate (SeO₄^2- 3.4 µM) or selenium methionine (SeMet 3.4 µM) on [Ca²⁺]_cp in pollen grains subjected to oxidative stress induced with H₂O₂ (0–5 mM), in the presence and absence of CaCl₂ in the incubation medium. Changes in cytosolic [Ca²⁺]_cp were assessed in control pollen grains exposed to SeO₄^2- or SeMet employed at a dose of 3.4 µM. The addition of SeO₄^2- or SeMet to the incubation medium was performed 50 s prior to the treatment with H₂O₂, after which fluorometric measurements were immediately started. CaCl₂ (1 mM) was included (right panel) to assess the extent of Ca²⁺ entry. Data are expressed as means ± SEM from four independent tests. In both panels, at any given concentration of H₂O₂, statistically significant differences are indicated by different letters, whereas identical letters highlight non-significant trends.

Figure 3

Dose response curve of SeO₄^2- or SeMet (0–10 µM) in [Ca²⁺]_cp during oxidative stress induced with 1.0 mM H₂O₂ in control pollen grains, in the absence (left panel) and presence (right panel) of CaCl₂ in the incubation medium. Data are expressed as means ± SEM from six independent tests. In both panels, at any given concentration of selenium, statistical significance of each set of data corresponding to a given dose of Se forms is indicated by different letters.

Figure 4

Effects of SeO₄^2- and SeMet on germination rates. The graph shows data obtained from control olive pollen grains incubated with increasing concentrations (0–100 µM) of SeO₄^2- (•) or SeMet (○). Results are expressed as % germination and represent the means ± SEM from five independent measurements, each of which supported by three technical replicates. Statistical significance of each set of data corresponding to a given dose of Se forms is indicated by different letters.

Figure 5

Germination rates under oxidative stress. The graph shows data obtained from control olive pollen grains pre-treated with increasing concentrations (0, 3.4, 10, and 50 µM) of SeO₄^2- (left panel) or Se-Met (right panel), subjected to oxidative stress induced with H₂O₂ (0, 1, and 5 mM). Results are reported as % of germinated pollen and expressed as means ± SEM from five independent tests, each of which included three technical replicates. Statistical significance of the data set is indicated by different letters.

Figure 6

Percentage germination of Se-enriched and control olive pollen grains under oxidative stress induced with H₂O₂ (0, 1, and 5 mM). Data are expressed as means ± SEM from five independent tests, each of which included three technical replicates. Statistical significance of each set of data corresponding to a given dose of H₂O₂ is indicated by different letters.