Mitigation of UV-B Radiation Stress in Tobacco Pollen by Expression of the Tardigrade Damage Suppressor Protein (Dsup).

Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation.

Assessing the effects of a commercial fungicide and an herbicide, alone and in combination, on Apis mellifera: Insights from biomarkers and cognitive analysis.

Agrochemicals play a vital role in protecting crops and enhancing agricultural production by reducing threats from pests, pathogens and weeds. The toxicological status of honey bees can be influenced by a number of factors, including pesticides. While extensive research has focused on the lethal and sublethal effects of insecticides on individual bees and colonies, it is important to recognise that fungicides and herbicides can also affect bees' health. Unfortunately, in the field, honey bees are exposed to mixtures of compounds rather than single substances. This study aimed to evaluate the effects of a commercial fungicide and a commercial herbicide, both individually and in combination, on honey bees. Mortality assays, biomarkers and learning and memory tests were performed, and the results were integrated to assess the toxicological status of honey bees. Neurotoxicity (acetylcholinesterase and carboxylesterase activities), detoxification and metabolic processes (glutathione S-transferase and alkaline phosphatase activities), immune system function (lysozyme activity and haemocytes count) and genotoxicity biomarkers (Nuclear Abnormalities assay) were assessed. The fungicide Sakura® was found to activate detoxification enzymes and affect alkaline phosphatase activity. The herbicide Elegant 2FD and the combination of both pesticides showed neurotoxic effects and induced detoxification processes. Exposure to the herbicide/fungicide mixture impaired learning and memory in honey bees. This study represents a significant advance in understanding the toxicological effects of commonly used commercial pesticides in agriculture and contributes to the development of effective strategies to mitigate their adverse effects on non-target insects.

Olive Leaf Extracts from Three Italian Olive Cultivars Exposed to Drought Stress Differentially Protect Cells against Oxidative Stress.

Olive leaves are an abundant by-product of olive oil production. Olive leaf extracts (OLEs) are rich in polyphenols, which can be used for health benefits. As polyphenols are the main antioxidant molecules in plants, plants typically increase their polyphenol content when exposed to drought stress. However, the phenolic profile of OLEs can vary in relation to the origin and variety of the plant material. In this work, olive leaf extracts from three different Italian olive cultivars (Giarraffa, Leccino, and Maurino) both exposed and not exposed to drought stress were studied in terms of antioxidant properties and profile, intestinal permeation, and protection against oxidative stress of human umbilical vein endothelial cells (HUVECs), since HUVECs are considered a model to study a wide range of diseases. OLEs from stressed Maurino and Giarraffa plants showed the highest increase in antioxidant capacity compared to controls. The phenolic profile of Maurino' was mainly increased by water deficit, with a large increase in the compounds oleuropein and luteolin-7-O-rutinoside. All tested extracts exposed to a water deficit protected HUVECs against oxidative stress by reducing ROS production, and this effect was more pronounced in OLEs from Giarraffa and Maurino exposed to drought stress compared to all other extracts. Finally, OLE from the stressed Giarraffa group showed a higher apparent permeability of antioxidant molecules than that of Maurino.

Pasta Enriched with Carrot and Olive Leaf Flour Retains High Levels of Accessible Bioactives after In Vitro Digestion.

The aim of this research was to evaluate the levels of antioxidants and polyphenols in pasta enriched with either carrot or olive leaf flours after simulating gastrointestinal digestion. Pasta samples were prepared with fixed amounts of carrot and olive leaf flours (15% and 6% of the total mixture, respectively). We measured the antioxidant capacity and polyphenol content at different stages of the pasta production process, starting from the initial flour to the cooked pasta, and tested samples of the liquid component and solid waste resulting from the digestion process. The antioxidant activity was measured by the FRAP method, while the polyphenol content was measured by the Folin-Ciocalteu method. Vitamin E contents were measured by HPLC. The pasta enriched with carrot (1.26 ± 0.05 mmol/100 g) and olive leaf (2.9 ± 0.07 mmol/100 g) exhibited higher antioxidant power compared to the unenriched pasta (0.8 ± 0.1 mmol/100 g). The polyphenol content followed a similar trend, with values of 131.23 ± 3.08 for olive flour-enriched pasta, 79.15 ± 1.11 for carrot flour-enriched pasta, and 67.5 ± 1.39 for the wheat-only pasta. The pasta samples maintained their antioxidant and polyphenol levels even after undergoing the simulated digestion process. Significantly, the liquid component of the pasta with olive leaf flours had the highest levels of antioxidants and polyphenols during all stages of the digestion process. According to the results of this study, pasta enriched with carrot and olive leaf flours shows promising potential for improving nutritional and functional properties by increasing antioxidant and polyphenol content. The samples were also evaluated by a sensory panel, which showed that fortification modified the perception of some organoleptic attributes without affecting the overall taste of the pasta.

Environmental Stress and Plants 2.0.

Following the success of our previous edition [...].

Tomato Biodiversity and Drought Tolerance: A Multilevel Review.

Ongoing global climate change suggests that crops will be exposed to environmental stresses that may affect their productivity, leading to possible global food shortages. Among these stresses, drought is the most important contributor to yield loss in global agriculture. Drought stress negatively affects various physiological, genetic, biochemical, and morphological characteristics of plants. Drought also causes pollen sterility and affects flower development, resulting in reduced seed production and fruit quality. Tomato (Solanum lycopersicum L.) is one of the most economically important crops in different parts of the world, including the Mediterranean region, and it is known that drought limits crop productivity, with economic consequences. Many different tomato cultivars are currently cultivated, and they differ in terms of genetic, biochemical, and physiological traits; as such, they represent a reservoir of potential candidates for coping with drought stress. This review aims to summarize the contribution of specific physio-molecular traits to drought tolerance and how they vary among tomato cultivars. At the genetic and proteomic level, genes encoding osmotins, dehydrins, aquaporins, and MAP kinases seem to improve the drought tolerance of tomato varieties. Genes encoding ROS-scavenging enzymes and chaperone proteins are also critical. In addition, proteins involved in sucrose and CO2 metabolism may increase tolerance. At the physiological level, plants improve drought tolerance by adjusting photosynthesis, modulating ABA, and pigment levels, and altering sugar metabolism. As a result, we underline that drought tolerance depends on the interaction of several mechanisms operating at different levels. Therefore, the selection of drought-tolerant cultivars must consider all these characteristics. In addition, we underline that cultivars may exhibit distinct, albeit overlapping, multilevel responses that allow differentiation of individual cultivars. Consequently, this review highlights the importance of tomato biodiversity for an efficient response to drought and for preserving fruit quality levels.

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.

Pollen-Pistil Interaction.

The aim of this Special Issue is to highlight the molecular dialogue between the pollen tube and the pistil [...].

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX.

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed "pollensomes", are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles.

Effects of UV-B Radiation on the Performance, Antioxidant Response and Protective Compounds of Hazelnut Pollen.

Increasing ultraviolet (UV) radiation is expected to become a problem in hazelnut cultivation. The aim of this study is to examine the effects of UV-B on hazelnut pollen. To this end, the pollens were exposed to UV-B for 1, 2, and 3 h at distances of 10, 20, 30, and 40 cm. Groups treated for 2 h at 20 cm and 3 h at 10 and 20 cm were identified as the most affected based on the results of viability, germination, and tube elongation. Further studies on these groups showed that UV-B does not change the DPPH radical scavenging activity for all groups. However, total phenolic compounds decreased after 3 h of treatment at 10 and 20 cm, while total flavonoid compounds decreased after all treatment groups. The UV-B absorbance of cytoplasmic and cell-wall-bound fractions decreased for all groups. The UV-B absorbance of the sporopollenin-derived fraction increased after 2 h of treatment at 20 cm but decreases after treatment for 3 h at 10 and 20 cm. In summary, exposure to UV-B for different times and distances adversely affected pollen grains in terms of pollen viability, germination rate, tube length, and the level of antioxidant molecules and UV-absorbing compounds.

Expression of Clementine Asp-Rich Proteins (CcASP-RICH) in Tobacco Plants Interferes with the Mechanism of Pollen Tube Growth.

Low-molecular-weight, aspartic-acid-rich proteins (ASP-RICH) have been assumed to be involved in the self-incompatibility process of clementine. The role of ASP-RICH is not known, but hypothetically they could sequester calcium ions (Ca2+) and affect Ca2+-dependent mechanisms. In this article, we analyzed the effects induced by clementine ASP-RICH proteins (CcASP-RICH) when expressed in the tobacco heterologous system, focusing on the male gametophyte. The aim was to gain insight into the mechanism of action of ASP-RICH in a well-known cellular system, i.e., the pollen tube. Pollen tubes of tobacco transgenic lines expressing CcASP-RICH were analyzed for Ca2+ distribution, ROS, proton gradient, as well as cytoskeleton and cell wall. CcASP-RICH modulated Ca2+ content and consequently affected cytoskeleton organization and the deposition of cell wall components. In turn, this affected the growth pattern of pollen tubes. Although the expression of CcASP-RICH did not exert a remarkable effect on the growth rate of pollen tubes, effects at the level of growth pattern suggest that the expression of ASP-RICH may exert a regulatory action on the mechanism of plant cell growth.

The legacy of kinesins in the pollen tube 30 years later.

The pollen tube is fundamental in the reproduction of seed plants. Particularly in angiosperms, we now have much information about how it grows, how it senses extracellular signals, and how it converts them into a directional growth mechanism. The expansion of the pollen tube is also related to dynamic cytoplasmic processes based on the cytoskeleton (such as polymerization/depolymerization of microtubules and actin filaments) or motor activity along with the two cytoskeletal systems and is dependent on motor proteins. While a considerable amount of information is available for the actomyosin system in the pollen tube, the role of microtubules in the transport of organelles or macromolecular structures is still quite uncertain despite that 30 years ago the first work on the presence of kinesins in the pollen tube was published. Since then, progress has been made in elucidating the role of kinesins in plant cells. However, their role within the pollen tube is still enigmatic. In this review, I will postulate some roles of kinesins in the pollen tube 30 years after their initial discovery based on information obtained in other plant cells in the meantime. The most concrete hypotheses predict that kinesins in the pollen tube enable the short movement of specific organelles or contribute to generative cell or sperm cell transport, as well as mediate specific steps in the process of endocytosis.

Environmental Stress and Plants.

Land plants are constantly subjected to multiple unfavorable or even adverse environmental conditions. Among them, abiotic stresses (such as salt, drought, heat, cold, heavy metals, ozone, UV radiation, and nutrient deficiencies) have detrimental effects on plant growth and productivity and are increasingly important considering the direct or indirect effects of climate change. Plants respond in many ways to abiotic stresses, from gene expression to physiology, from plant architecture to primary, and secondary metabolism. These complex changes allow plants to tolerate and/or adapt to adverse conditions. The complexity of plant response can be further influenced by the duration and intensity of stress, the plant genotype, the combination of different stresses, the exposed tissue and cell type, and the developmental stage at which plants perceive the stress. It is therefore important to understand more about how plants perceive stress conditions and how they respond and adapt (both in natural and anthropogenic environments). These concepts were the basis of the Special Issue that International Journal of Molecular Sciences expressly addressed to the relationship between environmental stresses and plants and that resulted in the publication of 5 reviews and 38 original research articles. The large participation of several authors and the good number of contributions testifies to the considerable interest that the topic currently receives in the plant science community, especially in the light of the foreseeable climate changes. Here, we briefly summarize the contributions included in the Special Issue, both original articles categorized by stress type and reviews that discuss more comprehensive responses to various stresses.

Distinct Tomato Cultivars Are Characterized by a Differential Pattern of Biochemical Responses to Drought Stress.

Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the damage caused by drought on crop plants and the mechanisms implemented to tolerate the stress. In this study, we focused on four cultivars of tomato, an economically important crop in the Mediterranean basin. We investigated the biochemical mechanisms of plant defense against drought by focusing on proteins specifically involved in this stress, such as osmotin, dehydrin, and aquaporin, and on proteins involved in the general stress response, such as HSP70 and cyclophilins. Since sugars are also known to act as osmoprotectants in plant cells, proteins involved in sugar metabolism (such as RuBisCO and sucrose synthase) were also analyzed. The results show crucial differences in biochemical behavior among the selected cultivars and highlight that the most tolerant tomato cultivars adopt quite specific biochemical strategies such as different accumulations of aquaporins and osmotins. The data set also suggests that RuBisCO isoforms and aquaporins can be used as markers of tolerance/susceptibility to drought stress and be used to select tomato cultivars within breeding programs.

Chemical Profiling of Two Italian Olea europaea (L.) Varieties Subjected to UV-B Stress.

The depletion of the stratospheric ozone layer due to natural and/or anthropogenic causes decreases the amount of UV-B radiation filtered, and consequently increases the risk of potential damage to organisms. In the Mediterranean region, high UV-B indices are frequent. Even for species typical of this region, such as the olive tree, the progressive increase in UV-B radiation represents a threat. This work aimed to understand how high UV-B radiation modulates the phenolic and lipophilic profile of olive varieties, and identify metabolites that enhance olive stress tolerance. Two Italian olive varieties were subjected to chronic UV-B stress, and leaves were analyzed by gas and liquid chromatography. The results indicated that the most representative phenolic and lipophilic compounds of Giarraffa and Olivastra Seggianese were readjusted in response to UV-B stress. The Giarraffa variety seemed better suited to prolonged UV-B stress, possibly due to the higher availability of flavonoids that could help control oxidative damage, and the accumulation of hydroxycinnamic acid derivatives that could provide strong UV-B shield protection. In addition, this variety contained higher levels of fatty acids (e.g., palmitic, α-linolenic, and stearic acids), which can help to maintain membrane integrity and accumulate more sorbitol (which may serve as an osmoprotectant or act a free-radical scavenger), terpenes, and long-chain alkanes, providing higher protection against UV-B stress.

Biochemical and cytological interactions between callose synthase and microtubules in the tobacco pollen tube.

KEY MESSAGE: The article concerns the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in the pollen tube. Results confirmed this association and immunogold labeling showed a colocalization. Callose is a cell wall polysaccharide involved in fundamental biological processes, from plant development to the response to abiotic and biotic stress. To gain insight into the deposition pattern of callose, it is important to know how the enzyme callose synthase is regulated through the interaction with the vesicle-cytoskeletal system. Actin filaments likely determine the long-range distribution of callose synthase through transport vesicles but the spatial/biochemical relationships between callose synthase and microtubules are poorly understood, although experimental evidence supports the association between callose synthase and tubulin. In this manuscript, we further investigated the association between callose synthase and microtubules through biochemical and ultrastructural analyses in the pollen tube model system, where callose is an essential component of the cell wall. Results by native 2-D electrophoresis, isolation of callose synthase complex and far-western blot confirmed that callose synthase is associated with tubulin and can therefore interface with cortical microtubules. In contrast, actin and sucrose synthase were not permanently associated with callose synthase. Immunogold labeling showed colocalization between the enzyme and microtubules, occasionally mediated by vesicles. Overall, the data indicate that pollen tube callose synthase exerts its activity in cooperation with the microtubular cytoskeleton.

Male Fertility under Environmental Stress: Do Polyamines Act as Pollen Tube Growth Protectants?

Although pollen structure and morphology evolved toward the optimization of stability and fertilization efficiency, its performance is affected by harsh environmental conditions, e.g., heat, cold, drought, pollutants, and other stressors. These phenomena are expected to increase in the coming years in relation to predicted environmental scenarios, contributing to a rapid increase in the interest of the scientific community in understanding the molecular and physiological responses implemented by male gametophyte to accomplish reproduction. Here, after a brief introduction summarizing the main events underlying pollen physiology with a focus on polyamine involvement in its development and germination, we review the main effects that environmental stresses can cause on pollen. We report the most relevant evidence in the literature underlying morphological, cytoskeletal, metabolic and signaling alterations involved in stress perception and response, focusing on the final stage of pollen life, i.e., from when it hydrates, to pollen tube growth and sperm cell transport, with these being the most sensitive to environmental changes. Finally, we hypothesize the molecular mechanisms through which polyamines, well-known molecules involved in plant development, stress response and adaptation, can exert a protective action against environmental stresses in pollen by decoding the essential steps and the intersection between polyamines and pollen tube growth mechanisms.

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.