Programmed Cell Death Reversal: Polyamines, Effectors of the U-Turn from the Program of Death in Helianthus tuberosus L.

This review describes a 50-year-long research study on the characteristics of Helianthus tuberosus L. tuber dormancy, its natural release and programmed cell death (PCD), as well as on the ability to change the PCD so as to return the tuber to a life program. The experimentation on the tuber over the years is due to its particular properties of being naturally deficient in polyamines (PAs) during dormancy and of immediately reacting to transplants by growing and synthesizing PAs. This review summarizes the research conducted in a unicum body. As in nature, the tuber tissue has to furnish its storage substances to grow vegetative buds, whereby its destiny is PCD. The review's main objective concerns data on PCD, the link with free and conjugated PAs and their capacity to switch the destiny of the tuber from a program of death to one of new life. PCD reversibility is an important biological challenge that is verified here but not reported in other experimental models. Important aspects of PA features are their capacity to change the cell functions from storage to meristematic ones and their involvement in amitosis and differentiation. Other roles reported here have also been confirmed in other plants. PAs exert multiple diverse roles, suggesting that they are not simply growth substances, as also further described in other plants.

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.

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.

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.

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.

Plant Transglutaminases: New Insights in Biochemistry, Genetics, and Physiology.

Transglutaminases (TGases) are calcium-dependent enzymes that catalyse an acyl-transfer reaction between primary amino groups and protein-bound Gln residues. They are widely distributed in nature, being found in vertebrates, invertebrates, microorganisms, and plants. TGases and their functionality have been less studied in plants than humans and animals. TGases are distributed in all plant organs, such as leaves, tubers, roots, flowers, buds, pollen, and various cell compartments, including chloroplasts, the cytoplasm, and the cell wall. Recent molecular, physiological, and biochemical evidence pointing to the role of TGases in plant biology and the mechanisms in which they are involved allows us to consider their role in processes such as photosynthesis, plant fertilisation, responses to biotic and abiotic stresses, and leaf senescence. In the present paper, an in-depth description of the biochemical characteristics and a bioinformatics comparison of plant TGases is provided. We also present the phylogenetic relationship, gene structure, and sequence alignment of TGase proteins in various plant species, not described elsewhere. Currently, our knowledge of these proteins in plants is still insufficient. Further research with the aim of identifying and describing the regulatory components of these enzymes and the processes regulated by them is needed.

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.

Insights into the Mechanisms of Heat Priming and Thermotolerance in Tobacco Pollen.

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca2+) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species.

Allergenic risk assessment of urban parks: Towards a standard index.

Allergenicity indices are a powerful tool to assess the health hazard posed by urban parks to pollen allergic subjects. Nonetheless, only few indices have been developed and applied to urban vegetation in the last decade, and they were never compared nor standardised over the same dataset. To address this issue, in this paper the two best-known allergenicity indices, the Urban Green Zones Allergenicity Index (IUGZA) and the Specific Allergenicity Index (SAI), have been calculated for the same park (the Botanical Garden of Bologna), collecting vegetation data through both systematic sampling and arboreal census. The results obtained with the two data collection methods were comparable for both indices, indicating systematic sampling as a reliable approximation of the total census. Besides, the allergenic risk resulted moderate to high according to SAI, and very low according to IUGZA. Since SAI does not consider the total volume of the vegetation, it was deemed less reliable than IUGZA in evaluating the allergenicity of an enclosed green space.

Monitoring techniques for pollen allergy risk assessment.

Understanding airborne pollen allergens trends is of great importance for the high prevalence and the socio-economic impact that pollen-related respiratory diseases have on a global scale. Pursuing this aim, aeropalynology evolved as a broad and complex field, that requires multidisciplinary knowledge covering the molecular identity of pollen allergens, the nature of allergen-bearing particles (pollen grains, pollen sub-particles, and small airborne particles), and the distribution of their sources. To estimate the health hazard that urban vegetation and atmospheric pollen concentrations pose to allergic subjects, it is pivotal to develop efficient and rapid monitoring systems and reliable allergic risk indices. Here, we review different pollen allergens monitoring approaches, classifying them into I) vegetation-based, II) pollen-based, and III) allergen-based, and underlining their advantages and limits. Finally, we discuss the outstanding issues and directions for future research that will further clarify our understanding of pollen aeroallergens dynamics and allergen avoidance strategies.

Pollen forecasting and its relevance in pollen allergen avoidance.

Pollinosis and allergic asthma are respiratory diseases of global relevance, heavily affecting the quality of life of allergic subjects. Since there is not a decisive cure yet, pollen allergic subjects need to avoid exposure to high pollen allergens concentrations. For this purpose, pollen forecasting is an essential tool that needs to be reliable and easily accessible. While forecasting methods are rapidly evolving towards more complex statistical and physical models, the use of simple and traditional methods is still preferred in routine predictions. In this review, we summarise and explain the main parameters considered when forecasting pollen, and classify the different forecasting methods in two groups: observation-based and process-based. Finally, we compare these approaches based on their usefulness to allergic patients, and discuss possible future developments of the field.

Treatment with spermidine alleviates the effects of concomitantly applied cold stress by modulating Ca2+, pH and ROS homeostasis, actin filament organization and cell wall deposition in pollen tubes of Camellia sinensis.

The aim of the current study was to examine the effect of spermidine treatment concomitant with cold stress on the elongation of Camellia sinensis pollen tube. When exogenous spermidine (0.05 mM) was applied concomitantly with cold stress, pollen germination rate and pollen tube length were significantly increased in comparison with cold stressed pollen tubes. In addition, spermidine treatment concomitantly with cold stress reduced pollen tube abnormalities induced by cold stress. Besides, cold-induced disorganizations of actin filaments were ameliorated after spermidine treatment along with cold stress because anisotropy levels of actin filaments in shank and apex of pollen tubes decreased. Changes in cold-induced callose distribution in the pollen tube cell wall were partially recovered after spermidine/cold stress treatment. Other cold-induced effects (decrease in Ca2+ content, reduction of pH gradient, accumulation of ROS) were reverted to adequate levels after spermidine treatment in conjunction with cold stress, indicating that pollen tubes are able to cope with stress. Thus, spermidine treatment reorganized the growth pattern of pollen tubes by modulating Ca2+ and ROS homeostasis, actin cytoskeleton organization, and cell wall deposition in Camellia sinensis pollen tubes under cold stress.

Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern.

In clementine, failure of fertilization can result in parthenocarpic fruit development, which has several advantages, such as seedless fruit, longer shelf-life, and greater consumer appeal. Recently, S-RNases have been identified in Citrus grandis, thus revealing that the self-incompatibility (SI) reaction relies on the S-RNase gametophytic mechanism. The fundamental role of environmental factors, mostly temperature, in determining the numbers of pollen tubes reaching the ovary is also well established in Citrus. In the present work, temperature-dependent pollen-pistil interactions in C. clementina were analyzed, focusing on several morphological aspects, as well as on polyamine (PA) content and the activity and distribution of transglutaminase (TGase), both reported to be involved in the SI response in pear and in pummelo. Results clearly indicate that temperature contributed to a different activation of the SI response, which occurs at optimal temperature of 25°C but was by-passed at 15°C. TGase activity was stimulated during the SI response, and it localized differently in the compatible and incompatible interaction: in compatible pollinated styles, TGase localized inside the style canal, while it was detected all around it in incompatible crosses. TGase localization and activity were congruent with the levels of soluble and insoluble conjugated PAs and with morphological evidences, which highlighted cell wall modification occurring as a result of SI.

A low molecular-weight cyclophilin localizes in different cell compartments of Pyrus communis pollen and is released in vitro under Ca2+ depletion.

Cyclophilins (CyPs) are ubiquitous proteins involved in a wide variety of processes including protein maturation and trafficking, receptor complex stabilization, apoptosis, receptor signaling, RNA processing, and spliceosome assembly. The ubiquitous presence is justified by their peptidyl-prolyl cis-trans isomerase (PPIase) activity, catalyzing the rotation of X-Pro peptide bonds from a cis to a trans conformation, a critical rate-limiting step in protein folding, as over 90% of proteins contain trans prolyl imide bonds. In Arabidopsis 35 CyPs involved in plant development have been reported, showing different subcellular localizations and tissue- and stage-specific expression. In the present work, we focused on the localization of CyPs in pear (Pyrus communis) pollen, a model system for studies on pollen tube elongation and on pollen-pistil self-incompatibility response. Fluorescent, confocal and immuno-electron microscopy showed that this protein is present in the cytoplasm, organelles and cell wall, as confirmed by protein fractionation. Moreover, an 18-kDa CyP isoform was specifically released extracellularly when pear pollen was incubated with the Ca2+ chelator EGTA.

Compatible and Incompatible Pollen-Styles Interaction in Pyrus communis L. Show Different Transglutaminase Features, Polyamine Pattern and Metabolomics Profiles.

Pollen-stigma interaction is a highly selective process, which leads to compatible or incompatible pollination, in the latter case, affecting quantitative and qualitative aspects of productivity in species of agronomic interest. While the genes and the corresponding protein partners involved in this highly specific pollen-stigma recognition have been studied, providing important insights into pollen-stigma recognition in self-incompatible (SI), many other factors involved in the SI response are not understood yet. This work concerns the study of transglutaminase (TGase), polyamines (PAs) pattern and metabolomic profiles following the pollination of Pyrus communis L. pistils with compatible and SI pollen in order to deepen their possible involvement in the reproduction of plants. Immunolocalization, abundance and activity of TGase as well as the content of free, soluble-conjugated and insoluble-bound PAs have been investigated. 1H NMR-profiling coupled with multivariate data treatment (PCA and PLS-DA) allowed to compare, for the first time, the metabolic patterns of not-pollinated and pollinated styles. Results clearly indicate that during the SI response TGase activity increases, resulting in the accumulation of PAs conjugated to hydroxycinnamic acids and other small molecules. Metabolomic analysis showed a remarkable differences between pollinated and not-pollinated styles, where, except for glucose, all the other metabolites where less concentrated. Moreover, styles pollinated with compatible pollen showed the highest amount of sucrose than SI pollinated ones, which, in turn, contained highest amount of all the other metabolites, including aromatic compounds, such as flavonoids and a cynnamoil derivative.

Immunoreactivity of Gluten-Sensitized Sera Toward Wheat, Rice, Corn, and Amaranth Flour Proteins Treated With Microbial Transglutaminase.

The aim of this study was to analyze the effects of microbial transglutaminase (mTG) on the immunoreactivity of wheat and gluten-free cereals flours to the sera of patients with celiac disease (CD) and non-celiac gluten sensitivity (NCGS). Both doughs and sourdoughs, the latter prepared by a two-step fermentation with Lactobacillus sanfranciscensis and Candida milleri, were studied. In order to evaluate the IgG-binding capacity toward the proteins of the studied flours, total protein as well as protein fractions enriched in albumins/globulins, prolamins and glutelins, were analyzed by SDS-PAGE and enzyme-linked immunosorbent assay (ELISA). Results showed that while mTG modified both gluten and gluten-free flour by increasing the amount of cross-linked proteins, it did not affect the serum's immune-recognition. In fact, no significant differences were observed in the immunoreactivity of sera from CD and NCGS patients toward wheat and gluten-free protein extracts after enzyme treatment, nor did this biotechnological treatment affect the immunoreactivity of control samples or the sera of healthy patients. These results suggest that mTG may be used as a tool to create innovative gluten and gluten-free products with improved structural properties, without increasing the immune-reactivity toward proteins present either in doughs or in sourdoughs.