Composition of Sexual Fluids in Cycas revoluta Ovules During Pollination and Fertilization.

The composition of fluids that mediate fertilization in cycads is described for the first time. Using tandem mass spectrometry, proteomes of two stages of fluid production, megagametophyte fluid and archegonial chamber fluid production, are compared in Cycas revoluta. These were compared with the proteome of another sexual fluid produced by ovules, the pollination drop proteins. Cycad ovules produce complex liquids immediately prior fertilization. Compared with the pollination drops that mainly had few proteins in classes involved in defense and carbohydrate modification, megagametophyte fluid and archegonial chamber fluid had larger proteomes with many more protein classes, e.g. proteins involved in programmed cell death. Using high-performance liquid chromatography, megagametophyte fluid and archegonial chamber fluid were shown to have elevated concentrations of smaller molecular weight molecules including glucose, pectin and glutamic acid. Compared to megagametophyte fluid, archegonial chamber fluid had elevated pH as well as higher osmolality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12229-021-09271-1.

Phylogenetic and functional signals in gymnosperm ovular secretions.

BACKGROUND AND AIMS: Gymnosperms are either wind-pollinated (anemophilous) or both wind- and insect-pollinated (ambophilous). Regardless of pollination mode, ovular secretions play a key role in pollen capture, germination and growth; they are likely also involved in pollinator reward. Little is known about the broad-scale diversity of ovular secretions across gymnosperms, and how these may relate to various reproductive functions. This study analyses the sugar and amino acid profiles of ovular secretions across a range of ambophilous (cycads and Gnetales) and anemophilous gymnosperms (conifers) to place them in an evolutionary context of their possible functions during reproduction. METHODS: Ovular secretions from 13 species representing all five main lineages of extant gymnosperms were sampled. High-performance liquid chromatography techniques were used to measure sugar and amino acid content. Multivariate statistics were applied to assess whether there are significant differences in the chemical profiles of anemophilous and ambophilous species. Data were compared with published chemical profiles of angiosperm nectar. Chemical profiles were placed in the context of phylogenetic relationships. KEY RESULTS: Total sugar concentrations were significantly higher in ovular secretions of ambophilous species than wind-pollinated taxa such as Pinaceae and Cupressophyta. Ambophilous species had lower amounts of total amino acids, and a higher proportion of non-protein amino acids compared with anemophilous lineages, and were also comparable to angiosperm nectar. Results suggest that early gymnosperms likely had ovular secretion profiles that were a mosaic of those associated with modern anemophilous and ambophilous species. Ginkgo, thought to be anemophilous, had a profile typical of ambophilous taxa, suggesting that insect pollination either exists in Gingko, but is undocumented, or that its ancestral populations were insect-pollinated. CONCLUSIONS: Chemical profiles of ovular secretions of ambophilous gymnosperms show a clear signal of pollinator-driven selection, including higher levels of carbohydrates than anemophilous taxa, lower levels of amino acids, and the presence of specific amino acids, such as ß-alanine, that are known to influence insect feeding behaviour and physiology.

Uptake and toxicity of glyphosate in the lichen Xanthoria parietina (L.) Th. Fr.

This study investigated if treatment of the lichen Xanthoria parietina (L.) Th. Fr. with glyphosate caused uptake of this herbicide as well as physiological alterations. Samples were treated with Glifene SL®, a common commercial glyphosate-based herbicide, at the lowest recommended doses (3.6g/L) as well as with doses slightly higher than the highest suggested (36 g/L). The results clearly showed glyphosate uptake in X. parietina proportionally to the dose provided. Adverse physiological effects were evident on the photosynthetic apparatus (photosynthetic efficiency, chlorophyll a content, chlorophyll degradation) as well as on the fungal respiration rates and cell membrane integrity (ergosterol content, dehydrogenase activity) already after 24h from treatment, also at the low application dose. It is concluded that lichens are suitable organisms for monitoring unwanted biological effects from the application of glyphosate-based herbicides, as well as for detecting the accumulation of this compound in the biota, thus screening for its environmental fate.

To each their own! Nectar plasticity within a flower mediates distinct ecological interactions.

Nuptial and extranuptial nectaries are involved in interactions with different animal functional groups. Nectar traits involved in pollination mutualisms are well known. However, we know little about those traits involved in other mutualisms, such as ant-plant interactions, especially when both types of nectaries are in the same plant organ, the flower. Here we investigated if when two types of nectaries are exploited by distinct functional groups of floral visitors, even being within the same plant organ, the nectar secreted presents distinct features that fit animal requirements. We compared nectar secretion dynamics, floral visitors and nectar chemical composition of both nuptial and extranuptial nectaries in natural populations of the liana Amphilophium mansoanum (Bignoniaceae). For that we characterized nectar sugar, amino acid and specialized metabolite composition by high-performance liquid chromatography. Nuptial nectaries were visited by three medium- and large-sized bee species and extranuptial nectaries were visited mainly by ants, but also by cockroaches, wasps and flies. Nuptial and extranuptial nectar differed regarding volume, concentration, milligrams of sugars per flower and secretion dynamics. Nuptial nectar was sucrose-dominated, with high amounts of γ-aminobutyric acid and ß-aminobutyric acid and with theophylline-like alkaloid, which were all exclusive of nuptial nectar. Whereas extranuptial nectar was hexose-rich, had a richer and less variable amino acid chemical profile, with high amounts of serine and alanine amino acids and with higher amounts of the specialized metabolite tyramine. The nectar traits from nuptial and extranuptial nectaries differ in energy amount and nutritional value, as well as in neuroactive specialized metabolites. These differences seem to match floral visitors' requirements, since they exclusively consume one of the two nectar types and may be exerting selective pressures on the composition of the respective resources of interest.

Brodifacoum Levels and Biomarkers in Coastal Fish Species following a Rodent Eradication in an Italian Marine Protected Area: Preliminary Results.

Brodifacoum is the most common rodenticide used for the eradication of invasive rodents from islands. It blocks the vitamin K cycle, resulting in hemorrhages in target mammals. Non-target species may be incidentally exposed to brodifacoum, including marine species. A case study conducted on the Italian Marine Protected Area of Tavolara Island was reported after a rodent eradication using the aerial broadcast of a brodifacoum pellet. Brodifacoum presence and effects on non-target marine organisms were investigated. Different fish species were sampled, and a set of analyses was conducted to determine vitamin K and vitamin K epoxide reductase concentrations, prothrombin time, and erythrocytic nuclear abnormalities (ENA) assay. In all the examined organisms, brodifacoum was not detected. The results obtained showed differences in vitamin K and vitamin K epoxide concentrations among the samples studied, with a positive correlation for three species between vitamin K, vitamin K epoxide, and fish weight. The prothrombin time assay showed a good blood clotting capacity in the fish. Higher abnormality values were recorded for four species. The results of this study suggest that it is possible to hypothesize that the sampled fish were not likely to have been exposed to brodifacoum and that consequently there are no negative issues concerning human consumption.

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir.

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

Foliar Application of Wood Distillate Alleviates Ozone-Induced Damage in Lettuce (Lactuca sativa L.).

This study examined whether foliar applications of wood distillate (WD) have a protective effect on photosynthesis and the antioxidant power of lettuce when exposed to an ecologically relevant O3 concentration. Seedlings of lettuce (Lactuca sativa L.) were fumigated daily with 60 ppb of O3 for 30 days, five hours per day. Once per week, 50% of the fumigated plants were treated with foliar applications of 0.2% WD, while control plants were treated with water. The results clearly showed the ability of WD to protect lettuce plants from ozone-induced damage. Specifically, WD-treated plants exhibited lower damage to the photosynthetic machinery, assessed through a series of chlorophyll fluorescence parameters, a higher chlorophyll content, higher antioxidant power, as well as antioxidant molecules, i.e., caffeic acid and quercetin, and higher biomass. Counteracting the overproduction of ozone-generated reactive oxygen species (ROS) is speculated to be the main mechanism by which WD protects the plant from ozone-induced damage.

Italian Tomato Cultivars under Drought Stress Show Different Content of Bioactives in Pulp and Peel of Fruits.

BACKGROUND: This study aims to evaluate the performance, in terms of accumulation of antioxidant compounds in fruits, of nine local and three commercial Italian tomato cultivars subjected to drought stress. The same local cultivars had been previously studied at morpho-physiological level. METHODS: The present manuscript analyzes drought stress as a tool to increase the amount of secondary metabolites that can enhance fruit quality. Nutraceutical characterization of the fruits was performed by analyzing the content of antioxidants, phenols, flavonoids, lycopene, ascorbic acid (vitamin C), rutin, caffeic acid, and naringenin. At the same time, plant sensitivity to stress during the reproductive phase was monitored in terms of flower abscission, fruit drop, and seed germination. RESULTS: Perina turns out to be the tomato cultivar with the best nutraceutical properties in the absence of stress while the Quarantino cultivar is so for flavonoid content (control plants) and lycopene and vitamin C content (stressed plants). Perina and Quarantino are the cultivars with the best response to drought and Perina has the highest concentrations of bioactives. Quarantino responds most effectively to stress in the reproductive phase. CONCLUSIONS: data confirm that drought stress increases bioactive production in some local cultivars of tomato, which produce higher quality fruits.

Bio-Based Solutions for Agriculture: Foliar Application of Wood Distillate Alone and in Combination with Other Plant-Derived Corroborants Results in Different Effects on Lettuce (Lactuca Sativa L.).

Bio-stimulants are showing growing success and are gradually replacing synthetic fertilizers in agriculture. Wood distillate (WD), also known as wood vinegar or pyroligneous acid, is a by-product of biomass pyrolysis and is increasingly used as a bio-stimulant for crop production. This study investigated whether weekly foliar applications of 0.25% and 0.50% WD have a differential effect on the chlorophyll and sugar content as well as biomass production in lettuce (Lactuca sativa L.). Moreover, the additional beneficial effect from the addition of corroborants of plant origin such as 3% soy lecithin and 5% flavonoid-rich wood glycolic extract to WD (BF) was investigated. Moreover, the possible toxicological concern from some potentially toxic elements (PTEs), namely Cd, Cu, Fe, Pb, and Zn, which may be abundant in WD was verified. After four weeks, we found that 0.25% WD not only increases lettuce biomass, which has an economic value, but also has beneficial effects on other qualitative parameters such as sugars and total sweetness. On the other hand, the use of 0.5% WD decreased the content of soluble sugars, suggesting a hormetic-type effect. We did not find evidence of further beneficial effects from the addition to WD of plant-derived corroborants, nor of any enrichment in the content of the investigated PTEs.

Evolutionary and Ecological Considerations on Nectar-Mediated Tripartite Interactions in Angiosperms and Their Relevance in the Mediterranean Basin.

The Mediterranean basin hosts a high diversity of plants and bees, and it is considered one of the world's biodiversity hotspots. Insect pollination, i.e., pollen transfer from male reproductive structures to conspecific female ones, was classically thought to be a mutualistic relationship that links these two groups of organisms, giving rise to an admirable and complex network of interactions. Although nectar is often involved in mediating these interactions, relatively little is known about modifications in its chemical traits during the evolution of plants. Here, we examine how the current sucrose-dominated floral nectar of most Mediterranean plants could have arisen in the course of evolution of angiosperms. The transition from hexose-rich to sucrose-rich nectar secretion was probably triggered by increasing temperature and aridity during the Cretaceous period, when most angiosperms were radiating. This transition may have opened new ecological niches for new groups of insects that were co-diversifying with angiosperms and for specific nectar-dwelling yeasts that originated later (i.e., Metschnikowiaceae). Our hypothesis embeds recent discoveries in nectar biology, such as the involvement of nectar microbiota and nectar secondary metabolites in shaping interactions with pollinators, and it suggests a complex, multifaceted ecological and evolutionary scenario that we are just beginning to discover.

Comparison of the Mineral and Nutraceutical Profiles of Elephant Garlic (Allium ampeloprasum L.) Grown in Organic and Conventional Fields of Valdichiana, a Traditional Cultivation Area of Tuscany, Italy.

In the Valdichiana area (Tuscany, Italy) an ancient native landrace of elephant garlic (Allium ampeloprasum L.), locally known as "Aglione della Valdichiana", has long been cultivated. The aim of this study was to check whether there are differences in the mineral and nutraceutical profiles of the Aglione della Valdichiana cultivated conventionally and organically. Based on the analysis by ICP-MS of a wide array of major, minor, essential, and non-essential trace elements as well as rare earth elements, and the evaluation of the content of polyphenols, flavonoids, antioxidants, soluble proteins, soluble sugars, and starch, as well as the weight and water content, it was concluded that differences in the mineral and nutraceutical profiles of organically and conventionally grown bulbs were very limited. Only a statistically (p < 0.05) higher concentration of Cd (+2620%), Co (+113%), Mn (+55%), Rb (+180%), and Sb (+180%), as well as glucose (+37%) in conventionally cultivated bulbs emerged. Cadmium was the only element slightly higher than in the "reference plant," but with a negligible risk (three orders of magnitude lower) for human health based on consumption. It is concluded that we failed to find evidence of healthier food or a higher nutraceutical quality for organically cultivated elephant garlic.

Modeling heavy metal release in the epiphytic lichen Evernia prunastri.

In this study, the release of Cu2+ and Zn2+ was investigated and modeled in the epiphytic lichen Evernia prunastri. Samples were incubated with solutions containing these metals at ecologically relevant concentrations (10 and 100 µM) and then transplanted to a remote area and retrieved after 1, 2, 3, 6, 12, and 18 months. The results showed that, after 12 months, all samples faced similar metal reductions of ca. 80-85%, but after this period, all the involved processes seem to be no longer capable of generating further reductions. These results suggest that the lichen E. prunastri can provide information about environmental improvements after exposure to high or very high pollution levels in a relatively short period of time.

Nutraceutical Characteristics of Ancient Malus x domestica Borkh. Fruits Recovered across Siena in Tuscany.

Background: A diet rich in fruits and vegetables contributes to lowering the risk of chronic diseases. The fruits of Malus x domestica are a rich dietary source of bioactive compounds, namely vitamins and antioxidants, with recognized action on human health protection. Tuscany is known for its rich plant biodiversity, especially represented by ancient varieties of fruit trees. Particularly noteworthy are the many ancient Tuscan varieties of apple trees. Methods: Sugar quantification via HPLC and spectrophotometric assays to quantify the antioxidant power and total polyphenol content revealed interesting differences in 17 old varieties of Malus x domestica Borkh. recovered in Siena (Tuscany). Results: The quantification of antioxidants, polyphenols, and the main free sugars revealed that their content in the old fruits was often superior to the widespread commercial counterparts ('Red Delicious' and 'Golden Delicious'). Such differences were, in certain cases, dramatic, with 8-fold higher values. Differences were also present for sugars and fibers (pectin). Most ancient fruits displayed low values of glucose and high contents of xylitol and pectin. Conclusions: The results reported here suggest the possible use of ancient apple varieties from Siena for nutraceutical purposes and draw attention to the valorization of local old varieties.

Pollination drop in Juniperus communis: response to deposited material.

BACKGROUND AND AIMS: The pollination drop is a liquid secretion produced by the ovule and exposed outside the micropyle. In many gymnosperms, pollen lands on the surface of the pollination drop, rehydrates and enters the ovule as the drop retracts. The objective of this work was to study the formation of the pollination drop in Juniperus communis, its carbohydrate composition and the response to deposition of conspecific pollen, foreign pollen and other particulate material, in an attempt to clarify the mechanism of pollination drop retraction. METHODS: Branches with female cones close to pollination drop secretion were collected. On the first day of pollination drop exposure, an eyelash mounted on a wooden stick with paraffin was used to collect pollen or silica gel particles, which were then deposited by contact with the drop. Volume changes in pollination drops were measured by using a stereomicroscope with a micrometer eyepiece 3 h after deposition. The volume of non-pollinated control drops was also recorded. On the first day of secretion, drops were also collected for sugar analysis by high-performance liquid chromatography. KEY RESULTS: The pollination drop persisted for about 12 d if not pollinated, and formed again after removal for up to four consecutive days. After pollination with viable conspecific pollen, the drop retracted quickly and did not form again. Partial withdrawal occurred after deposition of other biological and non-biological material. Fructose was the dominant sugar; glucose was also present but at a much lower percentage. CONCLUSIONS: Sugar analysis confirmed the general trend of fructose dominance in gymnosperm pollination drops. Complete pollination drop withdrawal appears to be triggered by a biochemical mechanism resulting from interaction between pollen and drop constituents. The results of particle deposition suggest the existence of a non-specific, particle-size-dependent mechanism that induces partial pollination drop withdrawal. These results suggest that the non-specific response may decrease the probability of pollen landing on the drop, reducing pollination efficiency.

Bioaccumulation, physiological and ultrastructural effects of glyphosate in the lichen Xanthoria parietina (L.) Th. Fr.

This study compared bioaccumulation as well as physiological (content of ergosterol as marker of fungal vitality and photosynthetic efficiency as marker of algal vitality) and ultrastructural effects of glyphosate in the lichen Xanthoria parietina, with the herbicide being provided either by soaking or spraying of the samples. The results showed that bioaccumulation of glyphosate is fast and proportional to the dose supplied, and is independent on the way it is supplied. Toxic effects resulting from glyphosate uptake by both soaking and spraying were evident at physiological and ultrastructural level, both in the algal and fungal partner, with negative effects being generally dose- and time-dependent. Since our results showed that the concentration of glyphosate bioaccumulated in X. parietina remains stable (at least up to 90 days), a perspective is open for using lichens in biomonitoring of atmospheric pollution by glyphosate as a side effect of the application of this herbicide.

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds.

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.

Determination of acrylamide in local and commercial cultivar of potatoes from biological farm.

This paper reports the results of a preliminary study on the characterization of parameters influencing formation of acrylamide in fried potatoes, from biological cultivation. The formation of acrylamide was investigated in relation to frying in biological extra virgin olive oil and commercial seed oil. Three different cultivars (Rossa di Colfiorito, Quarantina bianca genovese and Kennebec) were chosen. Asparagine, glucose, fructose and sucrose concentrations were determined in potato slice before frying, while acrylamide content was analysed by LC-ESI-MS/MS in the slices fried in seed and extra virgin olive oil. The Kennebec cultivar showed differences in its potential for acrylamide formation, which was primarily related to its relatively high asparagine and reducing sugars contents, respect the other local cultivars (particulary Quarantina). Values of acrylamide below detection limit (LOD) were found in Quarantina bianca genovese cultivar samples fried in extra virgin olive oil and peanuts seed oil and higher in peanuts seed oil fried potatoes of Kennebec cultivar.

Molecular relaxations in magnesium polymer electrolytes via GHz broadband electrical spectroscopy.

GHz broadband electrical spectroscopy (G-BES) is adopted to investigate the molecular relaxations and interactions occurring within the system in an oxygen- and water-free atmosphere in the 300 kHz-20 GHz and -40 to 250 °C frequency and temperature ranges, respectively. A new electrolyte for magnesium secondary batteries that can transfer magnesium ions efficiently is presented. This electrolyte is based on polyethylene glycol 400 and a polymeric form of δ-MgCl2 . The information obtained by G-BES is crucial for studying the conduction mechanism of these new electrolytes.