Carlo Alberto Palmerini

Inhalation of olive pollen (Olea europaea L.) is one of the main causes of allergy in Mediterranean countries and some areas of North America. The response to allergens consists in the production of inflammatory cytokines which is mediated by the deregulation of Ca²⁺ signals. In this study, the biological activity of the material released in olive pollen hydration (PMR) was tested on Ca²⁺ cytosolic of PE/CA-PJ15 cells (PJ-15). Ca²⁺ cytosolic was determined by fluorometric assay with the cell line PE/CA-PJ15 (PJ-15) labeled with the fluorescent probe FURA 2 AM. The material released in olive pollen hydration (PMR) was analyzed by HPLC for the determination of phenolic acids. PMR was subjected to fractionation by gel filtration, and the fractions with Ca²⁺-chelating activity were tested with SDS-PAGE and the single bands characterized by proteomic analysis. PMR showed high Ca²⁺-chelating activity and is able of blocking the increase Ca²⁺-cytosolic produced by thapsigargin (TG). PMR then restored Ca²⁺ homeostasis in PJ-15 cells deregulated by the endoplasmic reticulum Ca²⁺-ATPases inhibitor. It is therefore possible that PMR can antagonize the effects of allergens on Ca²⁺ cytosolic. The analytical characterization of the material released by the pollen highlighted in the pollen allergen Ole e 3 and in the p-coumaric acid the possible culprits of the Ca²⁺-antagonist activity of PMR. Furthermore, the sequence of Ole e 3 could provide information for the possible construction of a synthetic peptide to be used in an allergy-targeted Ca²⁺-antagonist therapy.

Climate change (CC), which causes temperatures to rise steadily, is causing global warming. Rising temperatures can reduce plant yield and affect pollen characteristics. In particular, heat stress strongly influences pollen viability for its sensitivity to this extreme environmental condition. This work evaluated the effect of heat stress on olive pollen after in vitro incubation at different temperatures (20, 30, and 40 °C). Furthermore, the potential of selenium-methionine (Se-met) in mitigating the detrimental effects of heat stress on olive pollen was investigated. In particular, how thermal stress can affect pollen was evaluated by testing the effect of temperature on pollen germinability and morphology and cytosolic Ca²⁺ content. The results suggest that the heat stress at 40 °C caused a marked reduction in the germination rate, changes in the morphology of the external pollen wall, and a decreased response to Ca²⁺-agonist agents. On the contrary, in vitro treatment of pollen with Se-met improved the germination rate and Ca²⁺-cytosolic homeostasis under heat stress conditions and confirmed the protective role of this compound in containing the hydrogen peroxide (H₂O₂) toxicity. Therefore, this study revealed that organic selenium could play a crucial role in promoting heat tolerance in olive tree pollen.

In this work, we studied the effects of in vitro oxidative stress applied by H₂O₂ to maize pollen germination and cytosolic Ca²⁺, taken as an experimental model to test the biological activity of extracts of emmer (Triticum turgidum L. spp. dicoccum (Schrank ex Shubler) Thell.) wheatgrass obtained from grains sprouted with distilled water, or salinity (50 mM) or selenium (45 mg L⁻¹ of Na₂SeO₃). Wheatgrass extracts were obtained in two ways: by direct extraction in methanol, which represented the free phenolic fraction of extracts (Ef), and by residual content after alkaline digestion, which made it possible to obtain extracts with the bound fraction (Eb). Comparative tests on maize pollen were carried out by differently combining H₂O₂ and either wheatgrass extracts or pure phenolic acids (4-HO benzoic, caffeic, p-coumaric and salicylic). The cytosolic Ca²⁺ of maize pollen was influenced by either H₂O₂ or pure phenolic acids or Ef, but not by Eb. The negative effect of H₂O₂ on maize pollen germination and cytosolic Ca²⁺ was mitigated by Ef and, slightly, by Eb. The extent of the biological response of Ef depended on the sprouting conditions (i.e., distilled water, salinity or selenium). The extracts of Se-biofortified wheatgrass were the most effective in counteracting the oxidative stress.

Selenium (Se) is an important micronutrient for living organisms, since it is involved in several physiological and metabolic processes. Biofortification with Se increases the nutritional and qualitative values of foods in Se-deficient regions and increases tolerance to oxidative stress in olive trees. Many studies have shown that Se, in addition to improving the qualitative and nutritional properties of EVO oil, also improves the plant’s response to abiotic stress. This study addressed this issue by monitoring the effects of Se on cytosolic Ca²⁺ and on the germination of olive pollen grains in oxidative stress. The olive trees subjected to treatment with Na-selenate in the field produced pollen with a Se content 6–8 times higher than the controls, even after 20 months from the treatment. Moreover, part of the micronutrient was organic in selenium methionine. The higher selenium content did not produce toxic effects in the pollen, rather it antagonized the undesirable effects of oxidative stress in the parameters under study. The persistence of the beneficial effects of selenium observed over time in pollens, in addition to bringing out an undisputed adaptability of olive trees to the micronutrient, suggested the opportunity to reduce the number of treatments in the field.

Selenium is an essential micronutrient that provides important benefits to plants and humans. At proper concentrations, selenium increases plant growth, pollen vitality, the shelf life of fresh products, and seems to improve stress resistance; these effects can certainly be attributed to its direct and indirect antioxidant capacity. For these reasons, in the present work, the effects of selenium at different dosages on in vitro cultivated olive explants were investigated to observe possible positive effects (in terms of growth and vigor) on the proliferation phase. The work was carried out on four different olive cultivars: “San Felice”, “Canino”, “Frantoio”, and “Moraiolo”. The explants were cultured in aseptic conditions on olive medium (OM), with the addition of 4 mg·L⁻¹ of zeatin, 30 g·L⁻¹ of sucrose, and 7 g·L⁻¹ of agar. The experimental scheme included a comparison between explants grown with five different concentrations of Na₂SeO₄ (0, 10, 20, 40, and 80 mg L⁻¹) added to the medium during three successive subcultures. Interesting information has emerged from the results and all varieties responded to different concentrations of Selenium. The optimal Se dosages varied for each cultivar, but in general, Se concentration between 10 and 40 mg L⁻¹ increased fresh and dry weight of the explants and shoot lengths. Se treatment induced in all cultivars and for all dosages used an increase in total Se content in proliferated explants. Furthermore, as the subcultures proceeded, the ability of the explants to absorb Se did not diminish. The Se content ranged from 8.55 to 114.21 µg kg⁻¹ plant DW in ‘Frantoio’, from 9.83 to 94.85 µg kg⁻¹ plant DW in ‘Moraiolo’, from 19.84 to 114.21 µg kg⁻¹ plant DW in ‘Canino’, and from 20.97 to 95.54 µg kg⁻¹ plant DW in ‘San Felice’. In general, the effect of selenium tends to decrease with the progress of subcultures and this suggests a sort of “adaptation” effect of the explants to its presence. The present study highlights for the first time the possibility of using in vitro cultures as biotechnological support to study supplementation with selenium and its effects on in vitro olive plant growth.

Wild sunflower (Helianthus annuus L.) is an invasive species widely distributed in several regions of the world, where it shares a large area with domesticated sunflower. The imidazolinone-tolerant sunflower enables the control of problematic weeds (such as Xanthium spp., Brassica spp., wild sunflower) with imidazolinone herbicides (Clearfield^® production system) in cultivated sunflower crops, but could facilitate the gene transfer of herbicide resistance, from cultivated sunflower to wild sunflower, generating hard-to-control weed biotypes or herbicide-resistant populations. The development of new practices that involve the selective inhibition of reproduction structures, such as pollen granules, could be an innovative strategy to minimize outcrossing and the origin of weed–crop hybrids in Clearfield^® production systems. In this study, the effects of mugwort (Artemisia vulgaris L.) aqueous extract on cytosolic Ca²⁺ and the germination of pollen grains collected from conventional, wild and IMI-tolerant sunflower were tested. The results showed that mugwort deregulated Ca²⁺ homeostasis and markedly reduced the germination of conventional and wild sunflower pollen, but not IMI-tolerant pollen. The HPLC analysis revealed the presence of phenolic acids belonging to the hydroxycinnamic and benzoic classes in the mugwort extract. Hydroxycinnamic acids (caffeic and ferulic) deregulated the cytosolic Ca²⁺ of conventional and wild sunflower pollen, but not those which were IMI-tolerant, similar to mugwort extract. Selective inhibition of wild sunflower pollen in the Clearfield^® sunflower crop contributes to a possible new weed management strategy, reducing the wild sunflower reproduction by seed, minimizing the potential risks of outcrossing with the formation of weed–crop hybrids. The Ca²⁺ selective chelating activity of caffeic or ferulic acids provides elements to be investigated for their possible use as an alternative to mugwort extract.

In this work, the biological activity of emmer (Triticum turgidum L. spp. dicoccum (Schrank ex Shubler) Thell.) wheatgrass extracts obtained from grains sprouted with distilled water, or salinity (50 mM) or selenium (45 mg L⁻¹ of Na₂SeO₃), was tested through an experimental biological model based on the germination and cytosolic Ca²⁺ homeostasis of maize pollen grains. The effects of thapsigargin (TG) and of four phenolic acids (PAs: ferulic, coumaric, salicylic and 3-HO benzoic) on maize pollen were also tested as controls. Wheatgrass extracts influenced both pollen cytosolic Ca²⁺ and germination. The Ca²⁺ agonist activity of emmer wheatgrass was transient, different from that of TG, which caused a depletion of the stored Ca²⁺ and a permanent alteration of Ca²⁺ homeostasis. The results obtained with extracts compared to those obtained with pure PAs suggest that PAs in unconjugated forms, which are known to be well represent in emmer wheatgrass, contribute to the biological activity of extracts. The extent of the biological response of emmer wheatgrass extracts was influenced by emmer sprouting conditions (i.e., distilled water, or salinity or selenium). Maize pollen treated with Se-enriched wheatgrass extracts showed a less perturbed cytosolic Ca²⁺ and a higher germination rate.

Olive is considered as a moderately salt tolerant plant, however, tolerance to salt appears to be cultivar-dependent and genotypic responses have not been extensively investigated. In this work, saline stress was induced in four olive cultivars: Arbequina, Koroneiki, Royal de Cazorla and Fadak 86. The plants were grown in 2.5 l pots containing 60% peat and 40% of pumice mixture for 240 days and were irrigated three times a week with half-strength Hoagland solution containing 0, 100 and 200 mM NaCl. The effects of salt stress on growth, physiological and biochemical parameters were determined after 180, 210, and 240 days of treatment. Saline stress response was evaluated in leaves by measuring the activity of GSH and CAT enzymatic activity, as well as proline levels, gas exchanges, leaves relative water content and chlorophyll content, and proline content. All the studied cultivars showed a decrease in Net Photosynthesis, leaves chlorophyll content and plant growth (mainly leaves dry weight) and an increase in the activity of GSH and CAT. In addition, the reduction of proline content in leaf tissues, induced an alteration of osmotic regulation. Among the studied cultivars Royal and Koroneiki better counteracting the effects of saline stress thanks to a higher activity of two antioxidant enzymes.