The Quest for Reliable Drought Stress Screening in Tetraploid Wheat (Triticum turgidum spp.) Seedlings: Why MDA Quantification after Treatment with 10% PEG-6000 Falls Short.

Drought stress poses significant productivity challenges to wheat. Several studies suggest that lower malondialdehyde (MDA) content may be a promising trait to identify drought-tolerant wheat genotypes. However, the optimal polyethylene glycol (PEG-6000) concentration for screening seedlings for drought tolerance based on MDA quantification is not clear. The aim of this study was to verify whether a 10% (w/v) PEG-6000 concentration-induced water stress was reliable for discriminating between twenty-two drought-susceptible and drought-tolerant tetraploid wheat (Triticum turgidum spp. durum, turanicum, and carthlicum) accessions based on MDA quantification. To do so, its correlation with morpho-physiological traits, notoriously related to seedling drought tolerance, i.e., Seedling Vigour Index and Seedling Water Content, was evaluated. Results showed that MDA content was not a reliable biomarker for drought tolerance, as it did not correlate significantly with the aforementioned morpho-physiological traits, which showed, on the contrary, high positive correlation with each other. Combining our study with the cited literature, it clearly emerges that different wheat genotypes have different "water stress thresholds", highlighting that using a 10% PEG-6000 concentration for screening wheat seedlings for drought tolerance based on MDA quantification is not reliable. Given the conflicting results in the literature, this study provides important insights for selecting appropriate methods for evaluating wheat seedling drought tolerance.

Identifying key environmental factors to model Alt a 1 airborne allergen presence and variation.

Fungal spores, commonly found in the atmosphere, can trigger important respiratory disorders. The glycoprotein Alt a 1 is the major allergen present in conidia of the genus Alternaria and has a high clinical relevance for people sensitized to fungi. Exposure to this allergen has been traditionally assessed by aerobiological spore counts, although this does not always offer an accurate estimate of airborne allergen load. This study aims to pinpoint the key factors that explain the presence and variation of Alt a 1 concentration in the atmosphere in order to establish exposure risk periods and improve forecasting models. Alternaria spores were sampled using a Hirst-type volumetric sampler over a five-year period. The allergenic fraction from the bioaerosol was collected using a low-volume cyclone sampler and Alt a 1 quantified by Enzyme-Linked ImmunoSorbent Assay. A cluster analysis was executed in order to group days with similar environmental features and then analyze days with the presence of the allergen in each of them. Subsequently, a quadratic discriminant analysis was performed to evaluate if the selected variables can predict days with high Alt a 1 load. The results indicate that higher temperatures and absolute humidity favor the presence of Alt a 1 in the atmosphere, while time of precipitation is related to days without allergen. Moreover, using the selected parameters, the quadratic discriminant analysis to predict days with allergen showed an accuracy rate between 67 % and 85 %. The mismatch between daily airborne concentration of Alternaria spores and allergen load can be explained by the greater contribution of medium-to-long distance transport of the allergen from the major emission sources as compared with spores. Results highlight the importance of conducting aeroallergen quantification studies together with spore counts to improve the forecasting models of allergy risk, especially for fungal spores.

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.

Pollen-Pistil Interaction.

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

Environment-oriented selection criteria to overcome controversies in breeding for drought resistance in wheat.

Wheat is one of the most important cereal crops, representing a fundamental source of calories and protein for the global human population. Drought stress (DS) is a widespread phenomenon, already affecting large wheat-growing areas worldwide, and a major threat for cereal productivity, resulting in consistent losses in average grain yield (GY). Climate change is projected to exacerbate DS incidence and severity by increasing temperatures and changing rainfall patterns. Estimating that wheat production has to substantially increase to guarantee food security to a demographically expanding human population, the need for breeding programs focused on improving wheat drought resistance is manifest. Drought occurrence, in terms of time of appearance, duration, frequency, and severity, along the plant's life cycle varies significantly among different environments and different agricultural years, making it difficult to identify reliable phenological, morphological, and functional traits to be used as effective breeding tools. The situation is further complicated by the presence of confounding factors, e.g., other concomitant abiotic stresses, in an open-field context. Consequently, the relationship between morpho-functional traits and GY under water deficit is often contradictory; moreover, controversies have emerged not only on which traits are to be preferred, but also on how one specific trait should be desired. In this review, we attempt to identify the possible causes of these disputes and propose the most suitable selection criteria in different target environments and, thus, the best trait combinations for breeders in different drought contexts. In fact, an environment-oriented approach could be a valuable solution to overcome controversies in identifying the proper selection criteria for improving wheat drought resistance.

Climate change effects on bread wheat phenology and grain quality: A case study in the north of Italy.

Increasing temperatures, heat waves, and reduction of annual precipitation are all the expressions of climate change (CC), strongly affecting bread wheat (Triticum aestivum L.) grain yield in Southern Europe. Being temperature the major driving force of plants' phenological development, these variations also have effects on wheat phenology, with possible consequences on grain quality, and gluten protein accumulation. Here, through a case study in the Bolognese Plain (North of Italy), we assessed the effects of CC in the area, the impacts on bread wheat phenological development, and the consequences on grain gluten quality. The increasing trend in mean annual air temperature in the area since 1952 was significant, with a breakpoint identified in 1989, rising from 12.7 to 14.1°C, accompanied by the signals of increasing aridity, i.e., increase in water table depth. Bread wheat phenological development was compared in two 15-year periods before and after the breakpoint, i.e., 1952-1966 (past period), and 2006-2020 (present period), the latest characterized by aridity and increased temperatures. A significant shortening of the chronological time necessary to reach the main phenological phases was observed for the present period compared to the past period, finally shortening the whole life cycle. This reduction, as well as the higher temperature regime, affected gluten accumulation during the grain-filling process, as emerged analyzing gluten composition in grain samples of the same variety harvested in the area both before and after the breakpoint in temperature. In particular, the proportion of gluten polymers (i.e., gliadins, high and low molecular weight glutenins, and their ratio) showed a strong and significant correlation with cumulative growing degree days (CGDDs) accumulated during the grain filling. Higher CGDD values during the period, typical of CC in Southern Europe, accounting for higher temperature and faster grain filling, correlated with gliadins, high molecular weight glutenins, and their proportion with low molecular weight glutenins. In summary, herein reported, data might contribute to assessing the effects of CC on wheat phenology and quality, representing a tool for both predictive purposes and decision supporting systems for farmers, as well as can guide future breeding choices for varietal innovation.

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.

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.

Morpho-Physiological Classification of Italian Tomato Cultivars (Solanum lycopersicum L.) According to Drought Tolerance during Vegetative and Reproductive Growth.

Irrigation is fundamental for agriculture but, as climate change becomes more persistent, there is a need to conserve water and use it more efficiently. It is therefore crucial to identify cultivars that can tolerate drought. For economically relevant crops, such as tomatoes, this purpose takes on an even more incisive role and local agrobiodiversity is a large genetic reservoir of promising cultivars. In this study, nine local Italian cultivars of tomatoes plus four widely used commercial cultivars were considered. These experienced about 20 d of drought, either at vegetative or reproductive phase. Various physio-morphological parameters were monitored, such as stomatal conductance (gs), photosynthesis (A), water use efficiency (WUE), growth (GI) and soil water content (SWC). The different responses and behaviors allowed to divide the cultivars into three groups: tolerant, susceptible, and intermediate. The classification was also confirmed by a principal component analysis (PCA). The study, in addition to deepening the knowledge of local Italian tomato cultivars, reveals how some cultivars perform better under stress condition than commercial ones. Moreover, the different behavior depends on the genotype and on the growth phase of plants. In fact, the Perina cultivar is the most tolerant during vegetative growth while the Quarantino cultivar is mostly tolerant at reproductive stage. The results suggest that selection of cultivars could lead to a more sustainable agriculture and less wasteful irrigation plans.

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.

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.

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.

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.

Cytoskeleton, Transglutaminase and Gametophytic Self-Incompatibility in the Malinae (Rosaceae).

Self-incompatibility (SI) is a complex process, one out of several mechanisms that prevent plants from self-fertilizing to maintain and increase the genetic variability. This process leads to the rejection of the male gametophyte and requires the co-participation of numerous molecules. Plants have evolved two distinct SI systems, the sporophytic (SSI) and the gametophytic (GSI) systems. The two SI systems are markedly characterized by different genes and proteins and each single system can also be divided into distinct subgroups; whatever the mechanism, the purpose is the same, i.e., to prevent self-fertilization. In Malinae, a subtribe in the Rosaceae family, i.e., Pyrus communis and Malus domestica, the GSI requires the production of female determinants, known as S-RNases, which penetrate the pollen tube to interact with the male determinants. Beyond this, the penetration of S-RNase into the pollen tube triggers a series of responses involving membrane proteins, such as phospholipases, intracellular variations of cytoplasmic Ca2+, production of reactive oxygen species (ROS) and altered enzymatic activities, such as that of transglutaminase (TGase). TGases are widespread enzymes that catalyze the post-translational conjugation of polyamines (PAs) to different protein targets and/or the cross-linking of substrate proteins leading to the formation of cross-linked products with high molecular mass. When actin and tubulin are the substrates, this destabilizes the cytoskeleton and inhibits the pollen-tube's growth process. In this review, we will summarize the current knowledge of the relationship between S-RNase penetration, TGase activity and cytoskeleton function during GSI in the Malinae.