CELLULAR BIOLOGY OF POLLEN

The mature pollen grains of angiosperms are composed of a relatively large vegetative cell with abundant material for pollen tube growth, and a small generative cell inside the vegetative cell where the two gametic cells are formed. These two cells are surrounded by a wall which protects them from mechanical and environmental damage. Before pollen liberation, rapid and dramatic dehydration occurs and the pollen volume is reduced to 35%, facilitating the dispersion of pollen grains by the wind. At this stage, the pollen has a stock of proteins available. After dispersion and pollination, the pollen grains undergo rehydration on the surface of the stigma. This hydration is achieved through stigmatic and pollen secretions. The pectin fraction of the intine pollen wall undergoes a rapid dilatation and gelation, and the specific proteins of the activation process are diffused. Many of these proteins are aeroallergens and are also involved in the allergenic process . A similar mechanism takes place when allergen-carrying pollen contacts the oral, nasal or eye mucosa. The allergens of pollen grains are then rapidly released thereby inducing the appearance of hay-fever symptoms in sensitised patients.

Cellular localization of pollen allergens in Gymnosperms and Angiosperms

The pollen allergens are intracellular soluble proteins, glycoproteins or lipoproteins of low molecular weight and are diffused through the pollen wall when the pollen comes in contact with the wet medium . The allergens are also located in the extra cellular sites, such as the intine, exine cavities and orbicules of Ubisch . Efforts to locate the sub cellular sites of pollen allergens were among the first reported applications of immunofluorescence techniques in plants. Studies applying a combination of transmission electron microscope and immunocytochemical methods located allergens inside cellular compartments: Bet v 1, Bet v 2, Lol p 1, Ole e 1 , Cry j 1 , Par j 1-Par j2 and allergen pools of Parietaria judaica , Zygophyllum fabago and poaceae  recognized by the IgE patients’ sera. All the studies have demonstrated the diffusion of these proteins in a wet medium. The major allergens of Platanus acerifolia pollen grains are located in the cistern of the endoplasmic reticulum and in the Golgi complex. The abundant production and elution of these proteins may alter the environment in which pollen tube elongation occurs, thus promoting a potential crosstalk between the pollen and the gynoecium . This relationship between the solubility of the pollen proteins and their allergenic potential is widely accepted .

Enzymatic activity associated with pollen allergens

The pollen hydration process, which takes place naturally on the stigma surface, is specifically designed to obtain success at the beginning of germination. Normally the soluble pollen proteins have enzymatic activity common in plant enzymes. The enzymatic activity of various pollen allergens is explained in more detail below.

Carbohydrates
The natural function of most pollen allergens remains largely unknown. A relatively small number of pollen allergens have been biochemically characterized as active enzymes. Most of these allergens have enzymatic activity related to carbohydrate metabolism, including: 1,3-β-glucanase of Ole e 9 , polygalacturonase of Cry j 2 , Cup a 1 , Pla a 2 a , and Phl p 13 , pectate lyase of Cry j 1 and invertase and pectin methyl esterase inhibitors of Pla a 1 .
Pollen grains require carbohydrates to succeed in the maturation process, so in mature pollen grains of Platanus acerifolia the cytoplasm contains abundant starch and other polysaccharide granules . Disturbances in sugar physiology in the anther lead to pollen abortion and subsequent sterility . The two major allergenic proteins of P. acerifolia (Pla a 1 and Pla a 2) have enzymatic activities related to carbohydrate metabolism. Our group located these major allergenic proteins in mature, hydrated and germinated pollen grains and established a new pattern of liberation and expression of these proteins which is related to sugar and pectin metabolisms .

Lipids
Plant cells contain proteins called lipid transfer proteins (LTPs) , which transfer phospholipids, glycolipids, fatty acids and sterols from liposomes or microsomes to mitochondria . Different roles have been suggested for the plant LTPs: cutin formation, embryogenesis, defence reactions against phytopathogens, symbiosis and the adaptation of plants to various environmental conditions . The two major allergens of Parietaria judaica, Par j 1 and Par j 2, belong to the LTP family . These LTPs, in the mature and hydrated pollen grains of P. judaica, participate in the secretory pathway, becoming available for pollen–stigma activity as well as inducing IgE antibody responses in allergic patients .

Profilins

Profilins are well-known ubiquitous cytoskeleton proteins with molecular masses of 12-15 kD, which play a essential role in regulating the activity in the microfilament system and signal transduction pathways . Their homology with the Bet v 2 pollen allergen of Betula has been demonstrated , and the IgE anti-profilin of Betula has been detected in polysensitized patients in regions without Betula trees . Profilins are highly cross-reactive allergens, which bind IgE antibodies of almost 20% of plant-allergic patients . Profilins are described as panallergens in a wide variety of plant sources and are responsible for allergy cross-reactions. Currently, there are numerous investigations of the cloning and sequencing of allergens, and many of them have a homology in the amino acid sequence with the profilin protein family . Plant profilins have been shown to be highly cross-reactive allergens that bind IgE antibodies of patients with food and tree pollen allergies .

Thaumatin-like proteins

Cup a 3 is a PR-5 protein derived from Cupressus arizonica pollen grains and has been recently cloned and sequenced . The increased expression of Cup a 3 is directly related to the pollution in the area where the pollen has been collected. The expression of this protein under polluted conditions has a direct incidence on the pollen allergenicity . Because of the homology sequences between PR-5 proteins and thaumatin, members of this family of proteins are referred to as thaumatin-like proteins (TLPs). TLPs are produced in response to pathogen infection or to osmotic stress .

Methods

Pollen grains have soluble proteins related with pollen-stigma recognition and are responsible for allergy responses. However, it is necessary to knowledge their functions and where the synthesis, storage and liberation occur.
Since the pollen grains are not static elements, the study should be planned in a sequential manner during their more active stages of development, hydration-activation and germination. The utilization of specific antibodies together with others which recognize panallergens, is an adequate strategy to deepen the knowledge of the dynamic of pollen proteins. This will bring new ideas offering simultaneous applications in two lines: 1) the fertilization mechanisms of plants with flowers (Gymnosperms and Angiosperms), and 2) the improvement in allergy diagnosis and in immunotherapy strategies.
The freezing protocols (link with Technical platform) for transmission electron microscopy (TEM), designed for a better observation of antigens in different tissues, are especially adequate for the detection of soluble proteins in pollen grains.
The pollen must be processed in vivo as quickly as possible, to preserve protein antigenicity as well as to obtain successful activation and germination. The process has various phases: to collect the pollen in its different stages of development, to process for TEM following freezing protocols, to obtain sections, and to label them with antibodies and to control the process by means of continuous TEM observations. The electron microphotographs evaluate the labelling of antigens using an image analysis program for an estimation of the frequency of gold particles associated with different cellular structures.

POLLEN AEROALLERGEN CONTROL IN THE ATMOSPHERE

Aerobiological surveillance of the atmosphere is a fundamental tool for pollinosis control. Pollinosis is one of the most annoying and persistent illnesses among non-lethal ones. Type I allergic disorders such as rhinoconjunctivitis, atopic eczema, and bronchial asthma are global problems afflicting up to 25% of the population in industrialized countries . A proper knowledge of local aeropalinology is crucial to establish correct etiological diagnostics . These studies have been carried out using light microscopy from the samples obtained with different sampling techniques, in order to count pollen and spores. Aerobiological studies are now being redirected due to the appearance of new information. 1) There is a rise in the morbidity of allergic diseases caused by pollen grains in urban areas of industrialized countries . 2) Many studies have shown that there is not always a high correlation between pollen counts and allergy symptoms. 3) Allergic particles which are smaller than pollen have been detected in the atmosphere before, during and after the pollination season . 4) These allergenic fractions, which range from 0.1 to 2.5 mm, are more efficient in order to penetrate the respiratory system . 5) The allergen load in the atmosphere appears as a new concept . All these facts pose a new challenge for aeropalinologists, as to achieve the pollen count in the samples, they must also determine the allergen load present in the atmosphere.
This allergen load should be correlated to clinical allergy symptoms, and this will show the presence of aeroallergens out of the blossom period. Allergens from dry pollen grains are cytoplasm proteins , but in gymnosperms, they are also situated in orbicules and walls . During hydration in the germination or isotonic mediums, these allergens elute within minutes . The same or similar biochemical processes can occur in the mucosa of sensitized patients and may induce allergic reactions . Although this mechanism might be responsible for symptoms of rhinoconjunctivitis, it cannot explain the occurrence of allergic asthma during or after the pollination period . Various sub-micronic particles may be responsible for allergic asthma, as intact pollen grains are unlikely to intrude into the deeper airways because of their size .
Different alternatives are under development to study the allergen load in the atmosphere. Among them immunoanalytical methods are found, which are widely used for indoor pollution studies but less developed for outside aerosol investigation. During the last two decades, various methods for measuring allergen exposure have been based on antibody allergen reaction systems as well as PCR analysis or surface plasmon resonance . The most essential information for pollinosis patients is the amount of airborne allergens and their temporary distribution .
Another aspect frequently found in aerobiology studies is that they tend to simulate the human respiratory system behaviour. The different methodologies described above use a wide variety of systems to obtain the aeroallergens. The majority of authors use low volume samplers starting at a sampling rate of 2 l/min and 28 l/min . However, high volume samplers are also used . Recently, the quantification of Ole e 1 and Par j 1-Par j 2 aeroallergens has been carried out using the low volume (16 l/min) Burkard cyclone sampler and the ELISA technique to compare the allergen load with the pollen count of Cartagena, a town in the Southeast of Spain .