Nicolas Visez

Patricia De Nadaï

Marie Choël
email: marie.choel@univ-lille.fr

Jinane Farah

Mona Hamzé

Hélène Sénéchal

Maxime Pauwels

Hélène Frérot

Michel Thibaudon

Pascal Poncet

Biochemical Composition of Phleum pratense Pollen Grains: a review

Keywords: timothy grass, Poaceae, allergy, asthma, proteins, allergens, lipids, pollen cytoplasmic granules

The Poaceae family is composed of 12 000 plant species. Some of these species produce highly allergenic anemophilous pollen grains (PGs). Phleum pratense pollen grains (PPPGs) emerged as a model for studies related to grass allergy. The biochemical composition of allergenic PGs has not yet been fully described despite potential health effects of PG constituents other than allergenic proteins. This review brings together the information available in literature aiming at creating a comprehensive picture of the current knowledge about composition of allergenic PGs from timothy grass. PPPGs have an average diameter between 30 to 35 µm and the mass of a single PG was reported between 11 and 26 ng. Pollen cytoplasm is filled with two types of pollen cytoplasmic granules (PCGs): the starch granules and the polysaccharide-particles (p-particles).

Starch granules have a size between 0.6 to 2.5 µm with an average diameter of 1.1 µm (estimated number of 1 000 granules per PG) while p-particles have a size ranging around 0.3 to 0.4 µm (estimated number between 61 000 to 230 000 p-particles per PG). Rupture of PG induce the release of PCGs and the dispersion of allergens in the inhalable fraction of atmospheric aerosol.

PPPGs are composed of sporopollenin, sugars, polysaccharides, starch, glycoproteins (including allergens), amino-acids, lipids, flavonoids (including isorhamnetin), diverse elements (the more abundant being Si, Mg and Ca), phenolic compounds, phytoprostanoids, carotenoids (pigments) metals and adsorbed pollutants. PPPG contains about a hundred different proteins with molecular masses ranging from 10 to 94 kDa, with isoelectric points from 3.5 to 10.6. Among these proteins, allergens are classified in eleven groups from 1 to 13 with allergens from groups 1 and 5 being the major contributors to Phl p pollen allergy. Major allergen Phl p 5 was quantified in PPPGs by several studies with concentration ranging from 2.7 and 3.5 µg.mg -1 in unpolluted environment. Values for other allergens are scarce in literature; only one quantitative assessment exists for allergen group Phl p 1, 2 and 4. The extractible lipidic fraction of PPPGs is estimated between 1.7 to 2.2% of the total PG mass. The main chemical families of lipids reported in PPPGs are: alkanes, alkenes, alcohols, saturated and unsaturated fatty acids, di-and trihydroxylated fatty acids, aldehydes and sterols. Several lipidic compounds with potential adjuvant effects on allergy have been specifically quantified in PPPGs: E2-like prostaglandin (PGE2), B4like leukotriene (LTB4), unsaturated fatty acids (linoleic and linolenic acids and their hydroxylated derivatives), adenosine, vitamins and phenolic compounds. Some other biochemical characteristics such as NAD(P)H oxidase, protease activity and pollen microbiome were described in the literature. The bioaccessibility in physiological conditions has not been described for most biochemicals transported by allergenic PPPGs. There is also a considerable lack of knowledge about the potential health effects of pollen constituents other than allergens. The variability of pollen composition remains also largely unknown despite its importance for plant reproduction and allergy in an environment characterized by chemical pollution, climate change and loss of biodiversity.

Introduction

The Poaceae family is composed of about 12 000 wind-pollinated species and their pollen grains (PGs) are considered as the major airborne biological threat to human health [START_REF] García-Mozo | Poaceae Pollen as the Leading Aeroallergen Worldwide: A Review[END_REF]. About 20% of the general population in the United States and Europe are sensitized to grass pollen allergens [START_REF] García-Mozo | Poaceae Pollen as the Leading Aeroallergen Worldwide: A Review[END_REF]. Main allergenic species for the Poaceae family are:

Alopecurus pratensis, Dactylis glomerata, Lolium perene, Cynodon Dactylon, Phleum pratense, and Poa annua [START_REF] Guerin | Pollen et Allergies[END_REF]. Timothy grass, Phleum Pratense L., is one among the Poaceae family and it is widely used in agriculture as a pasture and forage crop. Due to similarity and cross-reactivity of Phleum Pratense pollen grains (PPPGs) with those from other species from Poaceae, allergens extracted from PPPGs are used as a model in skin prick tests and PPPGs emerged as a model for studies related to allergy to grass pollen [START_REF] Andersson | Characteristics and Immunobiology of Grass Pollen Allergens[END_REF][START_REF] White | Key Pollen Allergens in North America[END_REF]. million pollen grains [START_REF] Albertine | Projected Carbon Dioxide to Increase Grass Pollen and Allergen Exposure Despite Higher Ozone Levels[END_REF] (photography provided by authors).

The main grass pollen season is observed in Europe from May to July [START_REF] Guerin | Pollen et Allergies[END_REF]. A single inflorescence of timothy grass can indeed produce up to 1.2 million PGs (Figure 1) [START_REF] Albertine | Projected Carbon Dioxide to Increase Grass Pollen and Allergen Exposure Despite Higher Ozone Levels[END_REF]. Atmospheric concentrations of grass pollen frequently reach more than a thousand of pollen grains per cubic meter [START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF]. Exposure to allergenic PGs is also effective in indoor air with persistence of allergens observed after the pollen season [START_REF] Dolecek | Dust from Carpeted and Smooth Floors. I. Comparative Measurements of Antigenic and Allergenic Proteins in Dust Vacuumed from Carpeted and Non-Carpeted Classrooms in Norwegian Schools[END_REF][START_REF] Evans | Predictors of group 5 grass-pollen allergens in settled house dust: comparison between pollination and nonpollination seasons[END_REF][START_REF] Evans | Predictors of group 5 grass-pollen allergens in settled house dust: comparison between pollination and nonpollination seasons[END_REF][START_REF] Holmquist | Quantification of Birch and Grass Pollen Allergens in Indoor Air[END_REF].

Pollen grains have historically been described as sole carriers of allergenic proteins as stated for example by [START_REF] Stull | The Allergically Active Substance in Pollen. a Chemical and Biologic Study of Phleum pratense (Timothy) Pollen[END_REF] « The allergen of timothy pollen is of protein nature and it is the only active substance in timothy pollen » [START_REF] Stull | The Allergically Active Substance in Pollen. a Chemical and Biologic Study of Phleum pratense (Timothy) Pollen[END_REF]. It is now clearly established that PGs and the pollen cytoplasmic granules (PCGs) are vectors of both allergenic proteins and other bioactive molecules [START_REF] Pointner | Initiating Pollen Sensitization -Complex Source, Complex Mechanisms[END_REF][START_REF] Suphioglu | Mechanism of Grass-Pollen-Induced Asthma[END_REF]. Pollen grains also transport lipids that are essential for plant reproductive functions [START_REF] Rejón | The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction[END_REF]. Lipids are also bioactive mediators capable of activating the cells of the immune system [START_REF] González Roldán | Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation[END_REF]. Other contributors to allergic inflammation also transported by PGs are pollen protease, NADPH oxidase and adsorbed gaseous or particulate pollutants [START_REF] Sénéchal | A Review of the Effects of Major Atmospheric Pollutants on Pollen Grains, Pollen Content and Allergenicity[END_REF][START_REF] Visez | Atmospheric Particulate Matter Adhesion onto Pollen: a Review[END_REF].

Despite sustained research efforts, the biochemical composition of allergenic PG is however not yet fully elucidated neither is the causal relationship between variation in PGs composition and pollen allergy risk (ie. the capacity of a PG, alone or with other factors such as air pollution, to induce health impact [START_REF] Galán | Recommended Terminology for Aerobiological Studies[END_REF]). First of all, research efforts are spread over different allergenic species, the most studied being for trees Betulaceae, Cupressaceae and Taxodiaceae, for grasses Lolium and Phleum and for weeds Ambrosia (ANSES, 2014). Pollen grains of allergenic species do share common characteristics but they have also many differences including first and foremost the nature and concentration of allergens. Secondly, the biochemical composition of pollen grains depends on both intrinsic and extrinsic factors. It may vary among species, but also among conspecific individuals, due to genetic determinants; it may also vary in space and time due to plant age or environmental variations (composition of soil, meteorological and climatic parameters…). Finally, the age of Anthropocene is characterized by significant levels of envrionmental perturbations, such as pollutions in soil, atmosphere, water. Such perturbations could influence the biochemical composition of plants and ultimately PGs. In an emblematic way, pollination period as well as airborne pollen concentrations are affected by climate change [START_REF] Besancenot | Changement climatique et pollinisation[END_REF][START_REF] Lake | Climate Change and Future Pollen Allergy in Europe[END_REF]. Increased concentrations of CO 2 may induce on certain species changes in production of pollen grains (increasing the amount of PGs) and allergenic protein contents [START_REF] Albertine | Projected Carbon Dioxide to Increase Grass Pollen and Allergen Exposure Despite Higher Ozone Levels[END_REF][START_REF] Kim | Does the Increase in Ambient CO 2 Concentration Elevate Allergy Risks Posed by Oak Pollen?[END_REF]. Interestingly, a more severe immunological response can also be induced by air pollution, either by direct effects on allergic patients or by alterations of PGs (directly on the PGs or indirectly via alterations of plants). For example, the worsening effects of soot particles and NO 2 are well established but the specific biochemical reactions and molecular processes responsible for their effects are still not clearly identified [START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF].

Determination of pollen biochemical composition is also important for estimating properties of pollen regarding condensation and glaciation nucleus and for estimating the role of PGs and their sub particles in the atmospheric water cycle [START_REF] Diehl | The Ice Nucleating Ability of Pollen: Part II. Laboratory Studies in Immersion and Contact Freezing Modes[END_REF][START_REF] Diehl | The Ice Nucleating Ability of Pollen: Part I: Laboratory Studies in Deposition and Condensation Freezing Modes[END_REF]. Moreover, a better description of biochemical composition of pollen is essential for understanding the biological mechanisms of respiratory allergic diseases and for the search of new therapeutic targets for the prevention and treatment of allergy. The past three decades have been fruitful in publications regarding PPPGs both on the characterization of allergens, lipids and other constituents. This review of the literature is in search of a detailed biochemical description of PPPGs.

Biochemical composition of Phleum pratense pollen grains

To the best of our knowledge, all biochemical compounds quantified in PPPGs and reported in literature are listed in

Diameter and mass of a single pollen grain

There is a general agreement in literature for an average diameter between 30 to 35 µm for PPPG (Table 2). In contrast, two ranges of values emerge from literature for the mass of a single PPPG: values between 11.1 and 16.4 ng.grain -1 on the one hand, and values between 23.7 and 26

ng.grain -1 on the other hand (Table 3). Those two sets may distinguish different cultivars with different diameters and/or densities, such as the WP Barpenta and the WP Rubato, for which reported masses were 16.4 ng.grain -1 and 23.7 ng.grain -1 respectively [START_REF] Jung | Grass Pollen Production and Group V Allergen Content of Agriculturally Relevant Species and Cultivars[END_REF]. Those two sets may indeed correspond to two varieties of Phleum with different diameters and/or densities. The historical value of Brown and Irving (13.7 ng) is the most frequently used in literature. In the absence of a consensus on the mass of a grain, this value was then used also in this review for the mass fraction calculations (conversion from ng.grain -1 to ng.mg -1 ). Settling velocity for PPPG has also been estimated in literature between 10 to 14 cm.s -1 [START_REF] Raynor | Dispersion and Deposition of Timothy Pollen from Experimental Sources[END_REF]. Average from different extraction methods [START_REF] Aloisi | Comparison of Extraction Methods for Poaceae Pollen Allergens[END_REF] Amino-acids 40

Extracted with aqueous solvent [START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF]

Internal lipids 26

Extracted with organic solvent (Farah et al., 2020a)

External lipids 22

Extracted with organic solvent (Farah et al., 2020b) Flavonoids 14.4

Extracted with organic solvent (Kolesnikov and Gins, 1999) Silicium (element) 7.3

Sum of free-, hydrolyzed-and tightly bound-Si [START_REF] Kolesnikov | Flavonoids and Silicon in Certain Plant Pollen[END_REF] Phenolic compounds 5

Extracted with organic solvent (Smiljanic et al., 13.7 [START_REF] Brown | The Size and Weight of Common Allergenic Pollens[END_REF] 16.4 Variety WP Barpenta [START_REF] Jung | Grass Pollen Production and Group V Allergen Content of Agriculturally Relevant Species and Cultivars[END_REF] 23.7 Variety WL Rubato [START_REF] Jung | Grass Pollen Production and Group V Allergen Content of Agriculturally Relevant Species and Cultivars[END_REF] 24±3 [START_REF] Murphy | Allelopathic Pollen Extract from Phleum pratense L. (Poaceae) Reduces Germination, In vitro, of Pollen of Sympatric Species[END_REF] 26 [START_REF] Guerin | Pollen et Allergies[END_REF] Pollen grain structure and sporopollenin Sporopollenin protects the plant gametes against a wide range of environmental attacks such as drying out and solar radiation [START_REF] Li | The Molecular Structure of Plant Sporopollenin[END_REF]. Despite its importance, the chemical structure of plant sporopollenin remained poorly documented and totally unexplored for Phleum pratense pollen [START_REF] Julier | Chemotaxonomy as a Tool for Interpreting the Cryptic Diversity of Poaceae Pollen[END_REF]. A cross-sectional view of a PPPG showing details of the pollen wall structure is presented in figure 2. The overall thickness of the pollen wall varies between 0.8 and 1.6 µm. In general, the pollen wall is composed of an outer envelope called exine (or exospore)

and an inner cellulosic membrane called intine (or endospore) [START_REF] Halbritter | Illustrated Pollen Terminology[END_REF]. The exine prevents the genetic material from damages due to pollen desiccation and is composed of two layers: the tectum and the foot layer, just above the intine [START_REF] Hesse | Pollen Terminology -An illustrated handbook[END_REF]. The tectum and the foot layer are separated by a region called the columella composed of reinforcing rods. The exine of PPPG is sculptured with spines (scabrae) and a microechinate ornamentation (Figure 3). The pollen pore allows shrinking (harmomegathy) and swelling of the grain caused by changes in moisture content. The circular aperture (pore) of PPPG has a lid (operculum).

Total proteins

The total amount of proteins extracted depends on the extraction protocol [START_REF] Aloisi | Comparison of Extraction Methods for Poaceae Pollen Allergens[END_REF].

Thus, when extraction was carried out with buffer lysis, by sonication with phosphate buffer saline solution (PBS) pH 7.5 or by continuous stirring with PBS, the total mass fraction of proteins on the same PPPG sample varied respectively from 45, 50 to 56 µg.mg -1 [START_REF] Aloisi | Comparison of Extraction Methods for Poaceae Pollen Allergens[END_REF]. These values are in perfect agreement with the determination of 51±4 µg.mg -1 carried out by others with an extraction by water with continuous agitation [START_REF] Rogerieux | Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O 3 , NO 2 , SO 2 )[END_REF]. Much lower values were subsequently determined at 13.2 µg.mg -1 by extraction with PBS at 4°C with constant agitation [START_REF] Schäppi | Concentrations of Major Grass Group 5 Allergens in Pollen Grains and Atmospheric Particles: Implications for Hay Fever and Allergic Asthma Sufferers Sensitized to Grass Pollen Allergens[END_REF] and at 26 µg.mg -1 by incubation at 37°C in vitro in phosphate-buffer pH 6.0 [START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF]. Those lower values could be tentatively explained by the lower temperature (4°C) in one case and by the absence of agitation in the other one (incubation). These differences may be explained also by the quantification methods used or by biological differences between samples.

Allergens

Allergens from the Poaceae family are one of the most frequent cause of allergic symptoms worldwide [START_REF] Mohapatra | Immunobiology of Grass Pollen Allergens[END_REF]. They are classified in eleven groups from 1 to 13 (groups 8 and 9 are absent) based on their structural and biological properties (table 4). Allergens from groups 1 and 5 (1a and b, and 5a and b) are the major contributors to Phl p pollen allergy [START_REF] Mohapatra | Immunobiology of Grass Pollen Allergens[END_REF]. PPPGs extract analyzed by 2D gel electrophoresis silver staining exhibits about 100 different proteins with molecular masses from 10 to 94 kDa and isoelectric points (pI) from 3.5 to 10.6 [START_REF] Abou Chakra | Proteomic Analysis of Major and Minor Allergens from Isolated Pollen Cytoplasmic Granules[END_REF][START_REF] Rogerieux | Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O 3 , NO 2 , SO 2 )[END_REF]. Other allergens such as the water-insoluble types have been studied in Phl p pollen and in PCG (Abou Chakra et al., 2011b). Water-insoluble allergens seem to play a role in the centrally mediated inflammatory response, whereas water-soluble allergens may be involved in the peripheral humoral response.

Table 4. Phleum pratense (timothy grass) pollen allergens. Prevalence data [START_REF] Mari | Skin Test with a Timothy Grass (Phleum pratense) Pollen Extract vs. IgE to a Timothy Extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: Epidemiological and Diagnostic Data[END_REF][START_REF] Niederberger | IgE Antibodies to Recombinant Pollen Allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) Account for a High Percentage of Grass Pollen-Specific IgE[END_REF]. IUIS: International Union of Immunological Societies.

Allergen Biochemical name MW /kDa Yes 83 [START_REF] Halim | Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Posttranslational Modifications[END_REF][START_REF] Laffer | Comparison of Recombinant Timothy Grass Pollen Allergens with Natural Extract for Diagnosis of Grass Pollen Allergy in Different Populations[END_REF][START_REF] Petersen | Post-Translational Modifications Influence IgE Reactivity to the Major Allergen Phl p 1 of Timothy Grass Pollen[END_REF][START_REF] Petersen | Studies on the Carbohydrate Moieties of the Timothy Grass Pollen Allergen Phl p I[END_REF][START_REF] Valenta | Diagnosis of Grass Pollen Allergy with Recombinant Timothy Grass (Phleum pratense) Pollen Allergens[END_REF][START_REF] Wicklein | Carbohydrate Moieties Can Induce Mediator Release: A Detailed Characterization of Two Major Timothy Grass Pollen Allergens[END_REF])

IUIS Phl p 2 Beta-expansin (fragment)
10-11 4.5-5.5

No 55 ( [START_REF] Barre | Homology Modeling of the Cellulose-Binding Domain of a Pollen Allergen from Rye Grass: Structural Basis for the Cellulose Recognition and Associated Allergenic Properties[END_REF][START_REF] Dolecek | Dust from Carpeted and Smooth Floors. I. Comparative Measurements of Antigenic and Allergenic Proteins in Dust Vacuumed from Carpeted and Non-Carpeted Classrooms in Norwegian Schools[END_REF][START_REF] Laffer | Comparison of Recombinant Timothy Grass Pollen Allergens with Natural Extract for Diagnosis of Grass Pollen Allergy in Different Populations[END_REF] Phl p 3

Beta-expansin (fragment) 10-11 8.9-9.3

No 37-70 [START_REF] Devanaboyina | High-Resolution Crystal Structure and Ige Recognition of the Major Grass Pollen Allergen Phl p 3[END_REF][START_REF] Petersen | Phl p 3: Structural and Immunological Characterization of a Major Allergen of Timothy Grass Pollen[END_REF][START_REF] Schweimer | Solution Structure of Phl p 3, a Major Allergen from Timothy Grass Pollen[END_REF]

IUIS

Phl p 4

Berberine bridge enzyme 59-67 9.0-10.5

Yes 70 [START_REF] Fischer | Characterization of Phl p 4, a Major Timothy Grass (Phleum pratense) Pollen Allergen[END_REF][START_REF] Leduc-Brodard | Characterization of Dac g 4, a Major Basic Allergen from Dactylis glomerata Pollen[END_REF][START_REF] Nandy | Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense)[END_REF][START_REF] Stumvoll | Purification, Structural and Immunological Characterization of a Timothy Grass (Phleum pratense) Pollen Allergen, Phl p 4, with Cross-Reactive Potential[END_REF][START_REF] Zafred | Crystal Structure and Immunologic Characterization of the Major Grass Pollen Allergen Phl p 4[END_REF]

IUIS

Phl p 5a

Phl p 5b Ribonuclease 25-34 5.0-8.5

Yes 50 [START_REF] Becker | Molecular Characterization of Timothy Grass Pollen Group V Allergens[END_REF][START_REF] Halim | Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Posttranslational Modifications[END_REF][START_REF] Laffer | Comparison of Recombinant Timothy Grass Pollen Allergens with Natural Extract for Diagnosis of Grass Pollen Allergy in Different Populations[END_REF][START_REF] Valenta | Diagnosis of Grass Pollen Allergy with Recombinant Timothy Grass (Phleum pratense) Pollen Allergens[END_REF] IUIS Phl p 6 Iso-flavone reductase 13 4.3-5.5

No 44 [START_REF] Blume | Microheterogeneity of the Major Grass Group 6 Allergen Phl p 6: Analysis by Mass Spectrometry[END_REF][START_REF] Petersen | Studies on the Carbohydrate Moieties of the Timothy Grass Pollen Allergen Phl p I[END_REF][START_REF] Vrtala | Molecular, Immunological, and Structural Characterization of Phl p 6, a Major Allergen and P-Particle-Associated Protein from Timothy Grass (Phleum pratense) Pollen[END_REF]) No 7 [START_REF] Niederberger | Calcium-Dependent Immunoglobulin E Recognition of the Apo-and Calcium-Bound Form of a Cross-Reactive Two Ef-Hand Timothy Grass Pollen Allergen, Phl p 7[END_REF][START_REF] Verdino | The Cross-Reactive Calcium-Binding Pollen Allergen, Phl p 7, Reveals a Novel Dimer Assembly[END_REF] Phl p 10 Cytochrome C 12 10-10.6

IUIS

No Unkown [START_REF] Ekramoddoullah | Determinants of Ryegrass Pollen Cytochrome c Recognized by Human Ige and Murine Monoclonal Antibodies[END_REF][START_REF] Ekramoddoullah | Allergenic Cross-Reactivity of Cytochromes c of Kentucky Bluegrass and Perennial Ryegrass Pollens[END_REF][START_REF] Sharma | Original Article. Molecular and Immunological Characterization of Cytochrome c: A Potential Cross-Reactive Allergen in Fungi and Grasses[END_REF]) Yes 25-75 (Suck et al., 2000a(Suck et al., , 2000b;;[START_REF] Wicklein | Carbohydrate Moieties Can Induce Mediator Release: A Detailed Characterization of Two Major Timothy Grass Pollen Allergens[END_REF] Quantification of allergens Phl p 5 was extensively quantified in PPPGs (Table 5); the values from the literature are in very good agreement. Values between 2.7 and 3.5 µg.mg -1 were reported for unpolluted PPPGs of various origins and over a time scale of about 20 years (first measurements in 1999). The amount of proteins extracted depends on the pH of the extraction medium: total proteins increased from 13.2 to 17.1 µg.mg -1 when the pH increased from 6.0 to 9.0 [START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF]. An average value of 3.2±0.5 µg.mg -1 was calculated for Phl p 5 from literature data mentioned with an asterisk in Table . PPPGs exposed to air pollution exhibit a lower mass concentration of Phl p 5 compared to unexposed pollens (table 5).

IUIS

Some information is available in literature on the quantification of allergens other than Phl p 5.

Allergens from group 2 were quantified at 1172 ng per 100 µg of total proteins [START_REF] Marth | Human Monoclonal Antibody-Based Quantification of Group 2 Grass Pollen Allergens[END_REF]. The Phl p 4 amount in pollen extract was determined at 0.2 µg.mg -1 quantified by an ELISA method. The absolute content of group 4 was in <1% relative to the total proteins of the Phl p pollen extract [START_REF] Fahlbusch | Detection and Quantification of Group 4 Allergens in Grass Pollen Extracts Using Monoclonal Antibodies[END_REF]. Moreover, the quantification of Phleum pratense allergens Phl p 1 and Phl p 5 in pollen extract was performed by mass spectrometry. In the extract Phl p 1b (Phl p 1 basic, 150 fmole.µL -1 ) was shown to be about 5 times less abundant than its variant Phl p 1a (Phl p 1 acid, 724 fmole.µL -1 ) and Phl p 5b (Phl p 5 basic, 4393 fmole.µL -1 ) was shown to be 9 times more abundant than the Phl p 5a (Phl p 5 acid, 499 fmole.µL -1 ) [START_REF] Seppälä | Absolute Quantification of Allergens from Complex Mixtures: A New Sensitive Tool for Standardization of Allergen Extracts for Specific Immunotherapy[END_REF]. The allergen concentration derived from this work (between 15-21 µg.mg -1 of Phl p 5) was not used for the calculation of the averaged Phl p 5 mass because of the discrepancy observed with other studies (average value of 3.2±0.5 µg.mg -1 ).

The amount of Phl p 5 in the atmosphere has a strong impact on the symptom load of allergenic patients [START_REF] Bastl | Can We Improve Pollen Season Definitions by Using the Symptom Load Index in Addition to Pollen Counts?[END_REF]. For the purpose of assessing individual exposure to allergens, pollen allergen potency (amount of allergen per mass of PG [START_REF] Galán | Recommended Terminology for Aerobiological Studies[END_REF]) are calculated for a given location by simultaneous analysis of concentration of pollen grains and of allergens [START_REF] Galán | Recommended Terminology for Aerobiological Studies[END_REF]. Comparison between the relative constancy of Phl p 5 per mg of pollen (or per pollen grain) and the pollen allergen potency reported in literature from grass species is questioning. Indeed, grass pollen allergen potency were reported as low as 0.2 µg.mg -1 and with a high variability from 0.05 to 2.3 µg.mg -1 [START_REF] Buters | Variation of the Group 5 Grass Pollen Allergen Content of Airborne Pollen in Relation to Geographic Location and Time in Season[END_REF][START_REF] Jochner | Seasonal Variation of Birch and Grass Pollen Loads and Allergen Release at Two Sites in the German Alps[END_REF][START_REF] Plaza | Mismatch in Aeroallergens and Airborne Grass Pollen Concentrations[END_REF].

Decrease of Phl p 5 release by pollution is a possible explanation as well as discrepancies between allergen content between grass species. Stored commercial pollen 0.9 [START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF] Pollen cytoplasmic granules (PCGs) and dispersion of allergens in respirable aerosols There are two types of pollen cytoplasmic granules (PCGs): the starch granules and the polysaccharide-particles (p-particles). PCGs were thoroughly investigated for their implications in pollen-related asthma.

Starch granules

Starch granules have a size between 0.6 to 2.5 µm with an average diameter of 1.1 µm (Abou Chakra et al., 2011a, 2011b;[START_REF] Suphioglu | Thunderstorm Asthma Due to Grass Pollen[END_REF]. Their number has been estimated to 1,000 starch granules per PPPG (Abou Chakra et al., 2011a). Starch granules were also been estimated between 700 and 1,000 per PG for ryegrass having a pollen size (and thus a volume) very close to the one of PPPG [START_REF] Singh | Isolation of cDNA Encoding a Newly Identified Major Allergenic Protein of Rye-Grass Pollen: Intracellular Targeting to the Amyloplast[END_REF][START_REF] Suphioglu | Thunderstorm Asthma Due to Grass Pollen[END_REF].

P-particles

P-particles have a size ranging around 0.3 to 0.4 µm [START_REF] Grote | Immunogold Electron Microscopic Localization of Timothy Grass (Phleum pratense) Pollen Major Allergens Phl p I and Phl p V after Anhydrous Fixation in Acrolein Vapor[END_REF][START_REF] Heslop-Harrison | The Growth of the Grass Pollen Tube: 1. Characteristics of the Polysaccharide Particles ("P-Particles") Associated with Apical Growth[END_REF]. The number of p-particles per pollen grains has not been directly measured. P-particles occupy between 20 to 30% of the PG volume corresponding to an estimate of 230,000 p-particles per PG (based on a spherical volume of pollen of 32 µm and spherical volume of p-particles of 0.35 µm) [START_REF] Heslop-Harrison | The Growth of the Grass Pollen Tube: 1. Characteristics of the Polysaccharide Particles ("P-Particles") Associated with Apical Growth[END_REF].

Arabinogalactan polysaccharides have been quantified to about 10% of the mass of PPPG [START_REF] Brecker | Structural and Immunological Properties of Arabinogalactan Polysaccharides from Pollen of Timothy Grass (Phleum pratense L.)[END_REF]. This polysaccharide fraction is composed by mass of 68% D-galactose and 32% L-arabinose. By considering that this mass of polysaccharide corresponds exclusively to pparticles, we obtain an estimate of 61,000 p-particles per pollen grain (density equals to 1.5 g.cm -3 and spherical particles of 0.35 µm diameter). We retain as an estimate between 61,000 and 230,000 of p-particles per pollen grain which is lower than the literature value of more than one million p-particles per PG [START_REF] Heslop-Harrison | The Growth of the Grass Pollen Tube: 1. Characteristics of the Polysaccharide Particles ("P-Particles") Associated with Apical Growth[END_REF]. Indeed, this value of one million would result in a mass of p-particles greater than that of a single pollen grain.

Localization of allergens

Visualization of allergen by immunogold-labelling technique has been widely used allowing localization of allergens between outer and inner parts of PPPGs. Allergens of group 1 and 5

were localized on exine and intine as well as in the cytoplasmic matrix between p-particles and cell organelles (mitochondria) [START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF][START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF][START_REF] Grote | Immunogold Electron Microscopic Localization of Timothy Grass (Phleum pratense) Pollen Major Allergens Phl p I and Phl p V after Anhydrous Fixation in Acrolein Vapor[END_REF]. Only allergens of group 5 were localized on amyloplasts (starch granules) [START_REF] Grote | Immunogold Electron Microscopic Localization of Timothy Grass (Phleum pratense) Pollen Major Allergens Phl p I and Phl p V after Anhydrous Fixation in Acrolein Vapor[END_REF]. Phl p 1 was found only in the intine [START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF]. Phl p 4 were localized in the exine, cytoplasm and amyloplast [START_REF] Fischer | Characterization of Phl p 4, a Major Timothy Grass (Phleum pratense) Pollen Allergen[END_REF]. Some allergens as Phl p 1 and Phl p 4 were also localized in pollen coat. (See below) (Bashir et al., 2013b;[START_REF] Rejón | The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction[END_REF].

In a comprehensive study, allergens Phl p 1, 4, 5, 6 and 12 were detected both on whole PPPGs and PCGs extracts whereas Phl p 11 was only detected on PCGs extract and Phl p 2 and 13 only on PGs extract [START_REF] Abou Chakra | Proteomic Analysis of Major and Minor Allergens from Isolated Pollen Cytoplasmic Granules[END_REF]. Phl p 6 has been localized on p-particles [START_REF] Vrtala | Molecular, Immunological, and Structural Characterization of Phl p 6, a Major Allergen and P-Particle-Associated Protein from Timothy Grass (Phleum pratense) Pollen[END_REF]. Confirming those localizations of allergens, PCGs are recognized by IgE from pollensensitive rat sera and they trigger lymph node proliferation in these rats [START_REF] Motta | Phleum pratense Pollen Starch Granules Induce Humoral and Cell-Mediated Immune Responses in a Rat Model of Allergy[END_REF].

Water soluble and water insoluble protein extracts from PCGs were recognized by IgE from rat sera and also from grass pollen allergic patient sera. The proteins recognized as allergens by IgE from sensitized rat may correspond to allergens also recognized by human IgE (Abou Chakra et al., 2011b). In a recent study, Phl p 2 and Phl p 5 are the allergens more concentrated in PPPG sub particles; Phl p 1 and Phl p 6 were also detected to a lesser extent [START_REF] Cecchi | Allergenicity at Component Level of Subpollens Particles from Different Sources Obtained by Osmolar Shock: A Molecular Approach to Thunderstorm-Related Asthma Outbreaks[END_REF].

The pollen coat also contains a number of proteins, most of which are also specifically synthesized in the tapetum layer. Many of these proteins are capable of triggering an IgEmediated immune response in humans, thus highlighting the importance of the pollen coat as also a source of aeroallergens (Bashir et al., 2013b). Among these allergens, Phl p 1, Phl p 4 and Phl p cystein protease were identified [START_REF] Rejón | The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction[END_REF].

Expulsion of PCGs from the cytoplasm

Expulsion of PCGs from the cytoplasm when pollen is immersed into an aqueous solution is a well-documented mechanism common to all grass species (figure 2) [START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF]. This cytoplasmic expulsion has been directly observed for PPPGs and it induces the release of hundreds of PCGs and allergens out of the PGs [START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF][START_REF] Swoboda | Molecular Characterization of Polygalacturonases as Grass Pollen-Specific Marker Allergens: Expulsion from Pollen via Submicronic Respirable Particles[END_REF][START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF]. Figure 2 shows PCGs expelled from the aperture of a PPPG immersed in water and a close-up view of oblong PCGs. Cytoplasmic expulsion occurs much less frequently when the time between pollen collection from grass and immersion is prolonged [START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF][START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF].

This expulsion of the cytoplasm is considered to be the major cause of allergen dispersion in the fine fraction of the atmospheric aerosol [START_REF] Suphioglu | Mechanism of Grass-Pollen-Induced Asthma[END_REF]. However, the mechanism by which allergens are transferred from the aqueous solution (in which the cytoplasm is expelled) to the small-sized respirable aerosol has never been studied. For this reason, work has been carried out to estimate the emission of fine particles charged with allergens directly from plant during pollination. An aerosol of particles smaller than 5 µm can be formed when grass and pollen is subjected to moisture-drying cycles and followed by exposure to an air flow [START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF].

This mechanism has been observed for grass species (Lolium and Cynodon) but not directly for Phleum. Other hypothesis for pollen rupture (not directly tested for PPPGs) are rupture induced by exposure to electrical fields and rupture induced by mechanical stress during wind transportation [START_REF] Vaidyanathan | Effects of Electric Fields on Pollen Rupture[END_REF][START_REF] Visez | Wind-Induced Mechanical Rupture of Birch Pollen: Potential Implications for Allergen Dispersal[END_REF]. Rupture of grass pollen species are directly linked to thunderstorm asthma [START_REF] Knox | Grass Pollen, Thunderstorms and Asthma[END_REF][START_REF] Suphioglu | Thunderstorm Asthma Due to Grass Pollen[END_REF][START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF].

Atmospheric pollution is a facilitating agent in inducing pollen rupture [START_REF] Motta | Traffic-Related Air Pollutants Induce the Release of Allergen-Containing Cytoplasmic Granules from Grass Pollen[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF].

Detection of allergens in the fine fraction of atmospheric aerosols PPPG allergens have been detected several times in the fine (respirable or less than 10 µm) fraction of atmospheric aerosol [START_REF] Schäppi | Analysis of Allergens in Ambient Aerosols: Comparison of Areas Subjected to Different Levels of Air Pollution[END_REF][START_REF] Spieksma | Seasonal Appearance of Grass Pollen Allergen in Natural, Pauci-Micronic Aerosol of Various Size Fractions. Relationship with Airborne Grass Pollen Concentration[END_REF][START_REF] Spieksma | Evidence of Grass-Pollen Allergenic Activity in the Smaller Micronic Atmospheric Aerosol Fraction[END_REF][START_REF] Taylor | Release of Allergens as Respirable Aerosols: a Link Between Grass Pollen and Asthma[END_REF]. However, Phl p 5 allergen was most of the time detected predominantly in coarse particle mode (PM>10 µm) [START_REF] Alan | The Effect of Geographical and Climatic Properties on Grass Pollen and Phl P 5 Allergen Release[END_REF][START_REF] Buters | Variation of the Group 5 Grass Pollen Allergen Content of Airborne Pollen in Relation to Geographic Location and Time in Season[END_REF]. After rainy events, grass pollen allergens were detected in the atmosphere while PGs were for their part not observed [START_REF] Holmquist | Quantification of Birch and Grass Pollen Allergens in Indoor Air[END_REF]. Large quantities of starch granules have also been directly observed in the atmosphere after rainy episodes but not on dry days [START_REF] Suphioglu | Thunderstorm Asthma Due to Grass Pollen[END_REF]. To the best of our knowledge, pparticles have never been directly observed in the atmosphere, probably because of their small size making their discrimination difficult among atmospheric particulate matter.

Lipidomic

Total lipidomic analysis Some lipids are bioactive compounds able to interfere in the cascade of biological reactions of the immune system. Some lipids can control early immune system response signals and amplify the response induced by allergens (Bashir et al., 2013a). This influence of lipids on immune response is of course directly ruled by their nature and biochemical characteristics.

Extractible lipidic fraction was determined as early as 1932 at 1.7% of the mass of PPPGs by Soxhlet extraction with an organic solvent [START_REF] Stull | The Allergically Active Substance in Pollen. a Chemical and Biologic Study of Phleum pratense (Timothy) Pollen[END_REF]. This value is very close to the one obtained by weighing of the dry extract (2.2%) obtained after pollen washing with methylene chloride (Farah et al., 2020b). However, use of organic solvents only allows to extract the external fraction of pollen lipids [START_REF] Evans | Predictors of group 5 grass-pollen allergens in settled house dust: comparison between pollination and nonpollination seasons[END_REF]. The internal fraction of PPPGs was recently obtained by grinding the grains prior to extraction: the total extractable lipids are distributed between 22 µg.mg -1 for the external fraction and 26 µg.mg -1 for the internal fraction (Farah et al., 2020a). Complete analysis of the extractable lipid fraction is still missing: less than 20% of lipids have been identified and quantified (Farah et al., 2020a(Farah et al., , 2020b)).

PPPGs from various geographical origins in the Czech Republic and the USA and from different harvest years (from 2007 to 2018) underwent lipid extraction with an organic solvent followed by analysis by gas chromatography coupled to mass spectrometry (Farah et al., 2020b). The variability in total lipid quantities was shown to be low between samples, less than 9%, despite geographical and pollen age differences. A larger dataset would be needed to confirm that (i) the lipids of pollen are stable despite storage and (ii) the lipid fraction is relatively constant in abundance in PPPGs from various geographical origins and years of collection.

Chemical composition of lipids

Main chemical families of lipids observed in PPPGs by extraction with an organic solvent are:

alkanes, alkenes, alcohols, saturated and unsaturated fatty acids, di-and tri-hydroxylated fatty acids and aldehydes (Bashir et al., 2013a;Farah et al., 2020b;[START_REF] González Roldán | Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation[END_REF].

Several sterols have also been detected but not quantified in extractable fraction of PPPGs (Bashir et al., 2013a;Farah et al., 2020b). The comparison between the semi-quantitative analysis carried out by Bashir et al. (2013) and the quantitative analyses of Farah et al. (2020) highlights significant disparities between the main chemical families despite similar procedures of extraction and analyses between studies (figure 4) (Bashir et al., 2013a;Farah et al., 2020aFarah et al., , 2020b). These differences are in our opinion due to differences in the interpretation of some mass spectra and/or due to simultaneous extraction of external and internal lipids consecutively to (uncontrolled) rupture of PGs during laboratory extraction protocols (Farah et al., 2020a(Farah et al., , 2020b)).

The external and internal lipid fractions of PPPGs share the same lipids but with a deeply different partitioning (figure 4Figure). A large predominance of saturated and unsaturated fatty acids has been observed in the cytoplasmic fraction (Farah et al., 2020a). The potential health effects of the presence of these specific lipids in PCGs of PPPGs has not been yet investigated.

Figure 4. Relative abundances of main chemical families of lipids from Phleum pratense pollen grains. Orange: external lipids (Bashir et al., 2013a); hatched orange: external lipids (Farah et al., 2020b); hatched blue: external lipids after exposure to ozone at 100 ppb for 16 hours (Farah et al., 2020b); grey: internal lipids (Farah et al., 2020a).

Prostaglandin and leukotriene

Pro-inflammatory substances, as E2-like prostaglandin (PGE2) and B4-like leukotriene (LTB4), have been extracted from PPPGs with aqueous solvent. Those so-called Pollen Associated Lipid Mediators (PALMs) were quantified in very low mass fractions of 0.00007 µg.mg -1 for PGE2 and of 0.00004 µg.mg -1 for LTB4 (Behrendt et al., 2001[START_REF] Behrendt | Timothy Grass (Phleum pratense L.) Pollen as Allergen Carriers and Initiators of an Allergic Response[END_REF][START_REF] Huss-Marp | Exposure of Grass Pollen to Volatile Organic Compounds Enhances Skin Prick Test Reactivity[END_REF]. Mass fractions of PALMs increased for PPPGs sampled in environment influenced by road-traffic compared to PPPGs sampled in rural environment (+100% for LTB4, +50% for PGE2) (Behrendt et al., 2001). The release of these substances occurred in less than 30 minutes after incubation of PPPGs in PBS (Behrendt et al., 2001).

Linoleic and linolenic acids and their derivatives

Discrepancies between studies exist for the quantification of linoleic and linolenic acids extracted from PPPGs: between 0.003 and 0.813 µg.mg -1 and between 0.051 and 5.0 µg.mg -1 respectively (Farah et al., 2020a(Farah et al., , 2020b;;[START_REF] Plötz | Chemotaxis and Activation of Human Peripheral Blood Eosinophils Induced by Pollen-Associated Lipid Mediators[END_REF][START_REF] Traidl-Hoffmann | Lipid Mediators from Pollen Act as Chemoattractants and Activators of Polymorphonuclear Granulocytes[END_REF]. Those differences could be explained, at least in part, by uncontrolled rupture of PPPGs during extraction procedure (Farah et al., 2020a). Mono-hydroxylated derivatives of linoleic acids and linolenic acid were quantified in lipids of PPPGs in the range between 0.006 and 0.010 µg.mg -1 [START_REF] Traidl-Hoffmann | Lipid Mediators from Pollen Act as Chemoattractants and Activators of Polymorphonuclear Granulocytes[END_REF].

Phytoprostanes and phytofurans

Phytoprostanoids are peroxidation products derived from α-linolenic acid and markers of oxidative stress among plants. Their concentrations in PPPGS was determined to 3.6 µg.mg -1 [START_REF] González Roldán | Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation[END_REF]. Phytoprostanes and phytofurans are rapidly released by PPPGs upon hydratation [START_REF] González Roldán | Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation[END_REF]. Phytoprostanoids bound to glycerol are able to trigger mast cells degranulation [START_REF] González Roldán | Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation[END_REF].

Adenosine

Adenosine was identified as a potent immunoregulatory substance in PPPGs and detected to mass fraction between 0.65 and 1.29 µg.mg -1 ; it is controversial whereas such low concentration of bioactive compound may or may not reach significant health-induced effects [START_REF] Gilles | Pollen Metabolome Analysis Reveals Adenosine as a Major Regulator of Dendritic Cell-Primed TH Cell Responses[END_REF][START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF].

Vitamins

PPPGS are rich in B9 vitamin (folic acid 1.1 µg.mg -1 ) and its conjugates (5.6 µg.mg -1 ) [START_REF] Nielsen | On the Occurrence of Folic Acid, Folic Acid Conjugates, and Folic Acid Conjugases in Pollen[END_REF]. Two other vitamins were also identified but not quantified in organic extracts of PPPGs: a-tocopherol and L-ascorbic acid (Bashir et al., 2013a).

Phenolic compounds

Phenolic compounds can provide an anti-oxidant capability to PPPGs by suppressing reactive oxidant response and by quenching free radicals [START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF]. The total phenol mass was estimated in PPPGs to 5 µg.mg -1 (gallic acid equivalent) and the mass of flavonoids to 14.4 µg.mg -1 [START_REF] Kolesnikov | Flavonoids and Silicon in Certain Plant Pollen[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF]. Isorhamnetin was identified in PPPGs and quantified to 3.1 µg.mg -1 [START_REF] Kolesnikov | Flavonoids and Silicon in Certain Plant Pollen[END_REF]. The flavonoids play a role in allergenicity of some proteins but its role on Phl p allergens was not yet described.

Water content

Release of proteins and allergens from PPPGs can be modified by an exposure of PGs to relative humidity [START_REF] Fritzsche | A Method for Investigating the Effects of Gaseous Pollutants on Pollen Ultrastructure and Allergen Release[END_REF]. There is however no measurement of water content specifically for PPPGs. The amount of water in pollen grains varies greatly at the time of pollination depending on the meteorological conditions: wind, humidity and temperature [START_REF] Heslop-Harrison | An Interpretation of the Hydrodynamics of Pollen[END_REF]. The water content of fresh pollen ranges from 15 to 35% for angiosperms but most PGs used in laboratory are dried to a water concentration of a few percents before storage [START_REF] Guerin | Pollen et Allergies[END_REF][START_REF] Heslop-Harrison | An Interpretation of the Hydrodynamics of Pollen[END_REF].

Others biochemical characteristics NAD(P)H oxidase

PPPGs and their extracts contained NAD(P)H oxidases [START_REF] Boldogh | Ros Generated by Pollen NADPH Oxidase Provide a Signal That Augments Antigen-Induced Allergic Airway Inflammation[END_REF] generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models [START_REF] Wang | NADPH Oxidase Activity in Allergenic Pollen Grains of Different Plant Species[END_REF]. ROS released by PGs has the ability to rapidly increase oxidative stress in respiratory system epithelium and as a consequence to augment allergic airway inflammation induced by PGs [START_REF] Boldogh | Ros Generated by Pollen NADPH Oxidase Provide a Signal That Augments Antigen-Induced Allergic Airway Inflammation[END_REF]. Pollen NAD(P)H oxidase was located at the surface and in the cytoplasm of pollen grains from species of the Poaceae family (not directly observed for PPPGs) [START_REF] Wang | NADPH Oxidase Activity in Allergenic Pollen Grains of Different Plant Species[END_REF].

Protease activity

Protease activity was observed for grass PGs (Secale cereale, Poa pratensis, Lolium perenne and Cynodon dactylon) but not directly for PPPGs [START_REF] Gunawan | Characterization of Proteases, Proteins, and Eicosanoid-Like Substances in Soluble Extracts from Allergenic Pollen Grains[END_REF][START_REF] Raftery | Mass Spectrometric Analysis of Electrophoretically Separated Allergens and Proteases in Grass Pollen Diffusates[END_REF].

Microbiome

DNA from bacteria and fungi was isolated from PPPGs. The microbial composition of PGs has been correlated with environmental exposure parameters. The microbial composition is also correlated with expression of allergens and quantities of PALMS in PPPGs [START_REF] Obersteiner | Pollen Associated Microbiome and Its Relationship to Pollution and Allergens[END_REF][START_REF] Obersteiner | Pollen-Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen[END_REF]. The potential effects of microbial stress on allergenicity of pollens are currently not well understood. Lipids originating from pollen microbiome may contribute to the sensitization process [START_REF] Bublin | Do Lipids Influence the Allergic Sensitization Process?[END_REF].

Pollutants

Several pollutants have been detected in PPPGs (Table 1) but they remain minor components on a mass basis of the whole pollen grains [START_REF] Chassard | Kinetic of NO 2 Uptake by Phleum Pratense Pollen: Chemical and Allergenic Implications[END_REF]Farah et al., 2020b;[START_REF] Goschnick | Surface and Depth Analysis of Pollen Treated with Atmospheric Trace Gases[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF]. PAHs and metals could be contributors to oxidative stress both onto pollen grains and on human cells after contact with a PPPG [START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF].

Discussion

Phleum pratense pollen grain (PPPG) is probably, with pollen from Betula and Ambrosia, one of the best described allergenic pollen grains. The biochemical description of PPPG was greatly improved during the last 30 years. Its main constituents have been very likely identified and some of them were quantified (Table 1). There is a growing evidence of the importance of molecules co-delivered with allergens during allergenic sensitization [START_REF] Pointner | Initiating Pollen Sensitization -Complex Source, Complex Mechanisms[END_REF]. PPPG is mainly composed of sporopollenin, oligosaccharides or polysaccharides, starch, proteins some of which are allergens, amino-acids, lipids and diverse elements, Si and Ca being the most abundant. Some important constituents of allergenic pollen like protease and NAD(P)H oxidase were not quantified in PPPGs. The way in which a priori incompatible substances (oxidants and antioxidants, protease and proteins) are distributed and segregated in pollen grains is also still rather poorly known (Taylor et al., 2007).

Data in table 1 could be considered as an average biochemical composition of PPPGs issued from various samples of pollen. The variability of pollen composition according to vegetal or environmental conditions remains largely unknown. Repeated quantification on a consequent number of samples is only available for Phl p 5 allergen: the agreement between studies is good with an average of 3.2±16% (table 4). On a limited number of samples, variability on lipids was estimated to 9% (Farah et al., 2020b). Most of others constituents quantified in PPPGs were determined for only one pollen sample (vitamins, extractible lipids, flavonoids, phenolic compounds…). A better knowledge of this variability is important for estimating the composition of PPPGs in the future in the context of a dramatic increase in atmospheric CO 2 concentration and of climate disasters. The effects of CO 2 on PPPG has been reported by one research group only for allergens and the number of PGs produced by anthers [START_REF] Albertine | Understanding the Links Between Human Health and Climate Change: Agricultural Productivity and Allergenic Pollen Production of Timothy Grass (Phleum pratense L.) Under Future Predicted Levels of Carbon Dioxide and Ozone[END_REF][START_REF] Albertine | Projected Carbon Dioxide to Increase Grass Pollen and Allergen Exposure Despite Higher Ozone Levels[END_REF]. Nothing is known about variability in lipids and other constituents nor about the adaptation of Phleum pratense to future climate change. Air pollution can also bring an important additional variability on certain biochemical parameters of PPPG: allergens release, changes in pollen composition, pollen rupture, oxidative alterations, adsorption of gaseous and particulate pollutants (Behrendt et al., 2001[START_REF] Behrendt | Allergens and Pollutants in the Air -a Complex Interaction[END_REF][START_REF] Behrendt | Localization, Release and Bioavailability of Pollen Allergens: The Influence of Environmental Factors[END_REF][START_REF] Chassard | Kinetic of NO 2 Uptake by Phleum Pratense Pollen: Chemical and Allergenic Implications[END_REF]Farah et al., 2020b;[START_REF] Goschnick | Surface and Depth Analysis of Pollen Treated with Atmospheric Trace Gases[END_REF][START_REF] Huss-Marp | Exposure of Grass Pollen to Volatile Organic Compounds Enhances Skin Prick Test Reactivity[END_REF][START_REF] Motta | Traffic-Related Air Pollutants Induce the Release of Allergen-Containing Cytoplasmic Granules from Grass Pollen[END_REF][START_REF] Risse | Health-Relevant Interaction Between Airborne Particulate Matter and Aeroallergens (Pollen)[END_REF][START_REF] Rogerieux | Modifications of Phleum pratense Grass Pollen Allergens following Artificial Exposure to Gaseous Air Pollutants (O 3 , NO 2 , SO 2 )[END_REF][START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF][START_REF] Smiljanic | In-Depth Quantitative Profiling of Post-Translational Modifications of Timothy Grass Pollen Allergome in Relation to Environmental Oxidative Stress[END_REF].

For a better knowledge of variability of pollen composition, the location and exact date of pollen harvesting should be systematically mentioned in published works. Lack of this information is a major gap in literature making it more difficult to discriminate potential effects linked to pollen ageing during storage or to specificity in composition linked to regional or seasonal variability. A striking example is the discrepancy in the mass of a single PPPG reported in various studies (table 3). It was also mentioned by Stull in 1932 that 10-year-old aqueous pollen extracts were less rich in protein than recently harvested pollen [START_REF] Stull | The Allergically Active Substance in Pollen. a Chemical and Biologic Study of Phleum pratense (Timothy) Pollen[END_REF]. Occurrence of pollen rupture drastically decreased after only several weeks of pollen storage [START_REF] Grote | Release of Allergen-Bearing Cytoplasm from Hydrated Pollen: a Mechanism Common to a Variety of Grass (Poaceae) Species Revealed by Electron Microscopy[END_REF]. A good practice for pollen-related publications would be to systematize the following information: date and place of harvest, storage conditions, defatted or not, supplier (if any) and water content.

PPPGs are rich in several biochemical compounds in concentrations ranging from 0.00004 to 384 µg.mg -1 (table 1). Human exposure to these compounds transported by pollen requires both their bioaccessibility (extraction from pollen in physiological conditions) and their bioactivity (inhibitory or promoting effect in the allergic reaction). Bioactivity presumes its bioaccessibility.

Bioaccessibility requires knowledge of the kinetic of release of the biochemical constituents of PG but also of their ability to diffuse into the mucus [START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF]. Such information on bioaccessibility remains scarce for PPPG constituents in particular and for allergenic pollens in general. Bioaccessibility also questions the distribution of biochemical constituents in the pollen grain structure and the state (intact or broken) of the PGs being inhaled. As example, the partitioning of lipids between the outer and cytoplasmic regions of PPPG has only been quantified in one study (Farah et al., 2020a). Rupture of PGs is a factor that facilitates the release of specific compounds as observed with lipids preferentially extracted by PBS on broken (ruptured) PPPGs (Farah et al., 2020a). However, this partitioning raises the question of the use of defatted pollens for the production of allergen extracts for skin prick tests. A pollen defatted with an organic solvent will always carry its internal lipids, which can also be released if the pollen is broken during an allergen extraction protocol.

Besides bioavailability, bioactivity requires that compounds extracted from PPPGs reach high enough concentrations for activities in physiological conditions. [START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF] have for example calculated concentrations of adenosine in nasal conditions based on assumptions on airborne pollen concentrations and of amount of nasal fluid. They conclude that, most of the time, nasal level of adenosine brought by PGs are too low to reach physiological levels [START_REF] Mueller | A Metabolomic, Geographic, and Seasonal Analysis of the Contribution of Pollen-Derived Adenosine to Allergic Sensitization[END_REF]. Their work illustrates the difficulties in estimating nasal concentrations of pollen constituents after their extraction in vivo conditions. To estimate bioactivities, there are clear lacks in our knowledge of both kinetic of pollen clearance and in the volume of diffusion of its constituents (i.e. physiological concentrations reached in close vicinity of deposited pollen in airways) [START_REF] Wang | NADPH Oxidase Activity in Allergenic Pollen Grains of Different Plant Species[END_REF].

Significant work has been carried out to characterize the eleven groups from 1 to 13 (groups 8 and 9 are absent) of allergens in PPPGs. However, only the major allergen Phl p 5 was repeatedly assayed with a mean value calculated at 3.2±0.5 µg.mg -1 . Phl p 5 and allergens from group 2 were the only allergens to be quantified in PPPGs and representing together 7-8% of total proteins.

The mass distribution of allergens between the different groups is not known, nor is the quantitative distribution of allergens between the outer and inner parts of the PG. Besides the 11 allergens described in Phleum pratense pollen ( The atmospheric concentrations of PGs do not necessarily coincide with the observations of symptom peaks [START_REF] Bastl | First Comparison of Symptom Data with Allergen Content (Bet v 1 and Phl p 5 Measurements) and Pollen Data from Four European Regions During 2009-2011[END_REF][START_REF] Jochner | Seasonal Variation of Birch and Grass Pollen Loads and Allergen Release at Two Sites in the German Alps[END_REF]. Furthermore, strong correlations were reported between symptom severity and air pollution but the biochemical mechanisms at work are largely unknown [START_REF] Berger | Impact of Air Pollution on Symptom Severity During the Birch, Grass and Ragweed Pollen Period in Vienna, Austria: Importance of O 3 in 2010-2018[END_REF][START_REF] Naclerio | International Expert Consensus on the Management of Allergic Rhinitis (AR) Aggravated by Air Pollutants: Impact of Air Pollution on Patients with AR: Current Knowledge and Future Strategies[END_REF][START_REF] Shiraiwa | Hazardous Components and Health Effects of Atmospheric Aerosol Particles: Reactive Oxygen Species, Soot, Polycyclic Aromatic Compounds and Allergenic Proteins[END_REF]. Biochemical alterations induced by pollution to PGs may be at least in part responsible of aggravation of symptoms in polluted environment. Establishing a relationship between symptom intensity and pollution-altered PGs is made difficult by the need to characterize the degree of pollution at the PG level with a time-resolved sampling. More generally, information about inhaled PGs are lacking: releasable quantities of allergens and bioactive compounds, water content, fraction of ruptured PGs, degree of alterations induced by pollution, number and nature of adhered atmospheric particles on the pollen surface… These biochemical parameters influence the biological micro-environment around a sedimented PG in human airways and contribute to modulate allergenic reactions.

Atmospheric pollen counts are considered relevant for public health and these samplings are widely used in routine across the world [START_REF] Lehtimäki | Aerobiology of Pollen and Pollen Antigens[END_REF]. The presence of allergens in the atmosphere in the absence of pollen grains and the direct link between symptoms and allergen load made several authors to conclude on the importance of additional allergen analysis in the atmosphere in addition to pollen counts [START_REF] Bastl | Can We Improve Pollen Season Definitions by Using the Symptom Load Index in Addition to Pollen Counts?[END_REF][START_REF] Buters | Variation of the Group 5 Grass Pollen Allergen Content of Airborne Pollen in Relation to Geographic Location and Time in Season[END_REF][START_REF] Jochner | Seasonal Variation of Birch and Grass Pollen Loads and Allergen Release at Two Sites in the German Alps[END_REF].

More generally, more information is needed about PGs (pollution, rupture of pollen grains) and sub-pollen particles (atmospheric concentrations) sampled directly in the atmosphere.

Conclusion

PPPGs are composed of polysaccharides, starch, allergenic and non-allergenic proteins, aminoacids, lipids, phenolic compounds, diverse elements (the more abundant being Si, Mg and Ca), phytoprostanoids, and adsorbed pollutants including metals and polycyclic aromatic hydrocarbons (PAH). The cytoplasm of one single PPPG is filled with about 1000 starch granules (average diameter 1.1 µm) and between 61,000 to 230,000 p-particles (0.3-0.4 µm). Much remains to be discovered about bioavailability and bioactivity of these constituents of PPPG and more generally of allergenic PGs. The lipid fraction of PPPG has, for example, not yet been fully described qualitatively and quantitatively with only 20% of extractible lipids being identified and quantified. Similarly, only 7-8% of total proteins have been quantified as allergens of group 2 and 5 allergen concentrations in other groups are unknown for PPPGs. The distribution of the constituents between the external and internal parts is also relatively poorly described in literature.

Phl p 5 is the only allergen that has been assayed by several research groups in PPPGs. A mean concentration of 3.2±0.5 µg.mg -1 has been determined for this allergen; this is the only quantitative estimation available for the variability of allergens over a heterogeneous set of PPPG samples. The variability of the biochemical composition of pollen is indeed not well known, including its determinants (vegetal, microbial, meteorological, geographical, or environmental causes). The existence of such variability could complicate our understanding of the biological effects of PPPG. Inspired on commercial batches of model particles used by atmospheric physicochemists, the aerobiology community should define a laboratory standard for PPPGs (and other allergenic PGs as well). This would require the establishment of sufficient stocks of pollen from plants grown under controlled, or at least known, conditions and storage of PPPGs under optimal conditions. This could allow intercomparative studies between laboratories and could hopefully generate new knowledge on PPPGs.

Figure 1 .

 1 Figure 1. (a) Inflorescence of timothy grass. (b) Close-up views of anthers. A single inflorescence can produce 1.2 million pollen grains (Albertine et al., 2014) (photography provided by authors).

Figure 2 .

 2 Figure 2. (left) Phleum pratense pollen grains in aqueous solution with internal and expelled pollen cytoplasmic granules (PCGs) and (right) close-up view of the cross-section of the pollen wall after glutaraldehyde fixation followed by osmium tetroxide treatment.

Figure 3 .

 3 Figure 3. SEM images of a Phleum pratense pollen grain showing (left) the sculpturing of the wall and (right) a close-up view of the single operculate aperture.

Table 1 .

 1 PPPGs are constituted of sugars, polysaccharides, starch,

	glycoproteins (including allergens), amino-acids, lipids, flavonoids (including isorhamnetin),
	diverse elements (the more abundant being Si, Mg and Ca), phenolic compounds,

phytoprostanoids, carotenoids (pigments) metals and adsorbed pollutants.

Table 1 .

 1 Current state of knowledge on biochemical constituents of Phleum pratense pollen grains. Abbreviations: PAHs polycyclic aromatic hydrocarbons, PALMS pollen-associated lipid mediators.

	Constituents	Mass	Details	References
		µg.mg -1
	Sugars	384	Fructose, sucrose, myo-inositol	(Mueller et al., 2016)
	P-particles	100-246	61000-230000 particles, spherical,	(Heslop-Harrison and Heslop-
			d=1.5 g.cm -3	Harrison, 1982)
	Starch granules	76	1000 granules, spherical, d=1.5	(Abou Chakra, 2009; Abou
			g.cm -3	Chakra et al., 2011a)
	Total proteins	50

Table 2 .

 2 Average diameter (µm) reported in literature for Phleum pratense pollen grains.

	2019)

Table 3 .

 3 Average mass (ng per pollen grain) reported in literature for Phleum pratense pollen grains.

	Mass of a single grain	Reference
	(ng per grain)
	11.1	(Abou Chakra, 2009)

Table 5 .

 5 Quantification of Phl p 5 in Phleum pratense pollen grains (*values used for our determination of average Phl p 5 release).

	Note

ACKNOWLEDGMENTS MC and NV thank the University of Lille and the Institut de Recherches Pluridisciplinaires en Sciences de l'Environnement (IREPSE Fed 4129) for financial support. The CaPPA project (Chemical and Physical Properties of the Atmosphere) is funded by the French National Research Agency (ANR) through the PIA (Programme d'Investissement d'Avenir) under contract ANR-11-LABX-005-01. This work is a contribution to the CPER research project CLIMIBIO.

The authors thank the French Ministère de l'Enseignement Supérieur et de la Recherche, the Hauts de France Region and the European Funds for Regional Economic Development for their financial support to this project.