Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

Seasonality of modern pollen and sediment deposition in an estuarine context: the Severn Estuary Levels, southwest England

Recent sedimentological and palynological research on subfossil Holocene banded sediments from the Severn Estuary Levels suggested seasonality of deposition, registered by variations in mineral grain-size and pollen assemblages between different parts of the bands. Here we provide data that strengthen this interpretation from sampling of modern sediments and pollen deposition on an active mudflat and saltmarsh on the margin of the Severn Estuary, and comparison with a vegetation survey and contemporary records of climate, river and tidal regimes. The results of grain-size analysis indicate deposition of comparatively coarse-grained silts during the relatively cool and windy conditions of winter and comparatively fine-grained sediments during relatively warm and calm summer months. Pollen analysis demonstrates the significance of long-term storage of pollen grains and fern spores in the estuarine waterbody, superimposed on which seasonal variations in pollen inputs from local and regional vegetation remain detectable. Copyright (C) 2007 John Wiley & Sons, Ltd.

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

Pollen ultrastructure in anther cultures of Datura innoxia. II. The generative-cell wall.

In young pollen grains of Datura innoxia, a wall of the usual hemispherical type separates the 2 gametophytic cells initially and, in the electron microscope, appears as an electron-translucent matrix which is contiguous with the intine. Before detachment of the generative cell from the intine, the matrix decreases in thickness and in places is dispersed altogether leaving the plasmalemmae on either side of it in close apposition. A particularly prominent zone, triangular in profile, is left where the wall joins with the intine. After detachment of the cell, remnants of the matrix can be seen distributed irregularly around the cell and it is supposed that these are partly derived from material in the triangular zone as the cell is drawn away from the intine. The wall residues persist throughout the maturation phase of the pollen and are considered to be either callose resulting from incomplete digestion of the initial wall, or some other polysaccharide material which is unevenly laid down along the wall and concentrated at the junction with the intine. In pollen induced into embryogenesis by anther culture, wall material is also distributed irregularly around the detached cell in a series of discrete zones, but these are more extensive than in vivo, closer together and in many instances highly dilated. The wall profiles thus have a beaded appearance, the 'beads' being connected together by short links of the 2 apposed plasmalemmae. The contents of the swollen zones have a similar electron density to that of the matrix in vivo but also show traces of a fibrillar component. It is postulated that this unusual swelling is a prelude to dispersal of the wall by disruption of the plasmalemmal links and to the establishment of cytoplasmic continuity between the 2 cells. The significance of such binucleate pollen grains in the formation of non-haploid embryos is discussed.

An improved methodology for the recovery of Zea mays and other large crop pollen, with implications for environmental archaeology in the Neotropics

We present a simple sieving methodology to aid the recovery of large cultigen pollen grains, such as maize (Zea mays L.), manioc (Manihot esculenta Crantz), and sweet potato (Ipomoea batatas L.), among others, for the detection of food production using fossil pollen analysis of lake sediments in the tropical Americas. The new methodology was tested on three large study lakes located next to known and/or excavated pre-Columbian archaeological sites in South and Central America. Five paired samples, one treated by sieving, the other prepared using standard methodology, were compared for each of the three sites. Using the new methodology, chemically digested sediment samples were passed through a 53 µm sieve, and the residue was retained, mounted in silicone oil, and counted for large cultigen pollen grains. The filtrate was mounted and analysed for pollen according to standard palynological procedures. Zea mays (L.) was recovered from the sediments of all three study lakes using the sieving technique, where no cultigen pollen had been previously recorded using the standard methodology. Confidence intervals demonstrate there is no significant difference in pollen assemblages between the sieved versus unsieved samples. Equal numbers of exotic Lycopodium spores added to both the filtrate and residue of the sieved samples allow for direct comparison of cultigen pollen abundance with the standard terrestrial pollen count. Our technique enables the isolation and rapid scanning for maize and other cultigen pollen in lake sediments, which, in conjunction with charcoal and pollen records, is key to determining land-use patterns and the environmental impact of pre-Columbian societies.

Anther and pollen development in some species of Poaceae (Poales)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pollen viability in Syagrus romanzoffiana and S-coronata (Arecaceae)

Palms of the Syagrus genus have several natural hybrids in Brazil. They are widely cultivated around the world as ornamental palm. S. romanzoffiana is the parental of natural hybrids with other Syagrus species that are potentially useful as palm cultivated for heart of palm production. The objective of this work was to evaluate the pollen grains viability got from plants grown in the Experimental Nursery of the FCAVJ/UNESP, Jaboticabal, SP, Brazil. Pollen grains were sampled from inflorescence in three occasions: in the right day of anthesis, one day before and one day after anthesis. For each day, the inflorescence was subdivided in epical, medium and basal portions where pollen grains were collected. The results shown that: a) for S. coronata there was no statistical differences in pollen viability (pollen viability average = 92.89%); b) for S. romanzoffiana the average pollen viability was 87.27%, 87.47% e 86.85% before, during and after the anthesis, respectively. The basal portion pollen grains had lower viability (85.19% - p<0.01) than those from the medium (88.17%) and the apical (88.46%) portion of the inflorescence. These results shown that both species have no problems with pollen viability and can be used as parental of hybrids with other Syagrus species.

Development and cytology of pollen in Dahlstedtia Malme (Leguminosae : Papilionoideae)

Microsporogenesis, chromosome number, meiotic behaviour and meiotic index were investigated in Dahlstedtia pinnata and D. pentaphylla, two legume species occurring largely in Brazil, in order to ascertain whether the pollen could limit fertilization events. Archesporial cells originate primary sporogenous and anther wall precursor cells, the tapetum is uniseriate, uninucleate and glandular. Tetrads are tetrahedric or decussate, and cytokinesis is of the simultaneous type. Mature pollen grains are tricolpate and bicellular. No abnormalities in microsporogenesis were found. In both species the chromosome number is n = 11, a number not reported previously. The base number for Dahlstedtia is also 11, because cytological observations include both species of Dahlstedtia. D. pentaphylla has a higher meiotic index and lower individual variation values, and it is considered meiotically stable. Its pollen grains do not limit fertilization. D. pinnata has a lower meiotic index, and the pollen is one of the factors which limit fertilization. Furthermore, D. pinnata has numerous adventitious shoots, which suggest that vegetative propagation is important in its reproductive process. (C) 2002 the Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 138, 461-471.

Pollen viability in Syagrus romanzoffiana and S. coronata (Arecaceae)

Palms of the Syagrus genus have several natural hybrids in Brazil. They are widely cultivated around the world as ornamental palm. S. romanzqffiana is the parental of natural hybrids with other Syagrus species that are potentially useful as palm cultivated for heart of palm production. The objective of this work was to evaluate the pollen grains viability got from plants grown in the Experimental Nursery of the FCAVJ/UNESP, Jaboticabal, SP, Brazil. Pollen grains were sampled from inflorescence in three occasions: in the right day of anthesis, one day before and one day after anthesis. For each day, the inflorescence was subdivided in apical, medium and basal portions where pollen grains were collected. The results shown that: a) for S. coronata there was no statistical differences in pollen viability (pollen viability average = 92.89%); b) for S. romanzoffiana the average pollen viability was 87.27%, 87.47% e 86.85% before, during and after the anthesis, respectively. The basal portion pollen grains had lower viability (85.19% - p<0.01) than those from the medium (88.17%) and the apical (88.46%) portion of the inflorescence. These results shown that both species have no problems with pollen viability and can be used as parental of hybrids with other Syagrus species.

Protein requirements in larvae and adults of Scaptotrigona postica (Hymenoptera: Apidia, Meliponinae): Midgut proteolytic activity and pollen digestion

The number and degree of digestion of pollen grains in the midgut and rectum, the midgut proteolytic activity and the time of pollen grain passage through the digestive tract in the stingless bee Scaptotrigona postica (Latreille) have been analyzed. The results show similar protein requirements among larvae, nurse bees and queens, as well as between forager bees and old males, but these requirements are higher in individuals from the former groups than in those from the latter. Although protein requirements have been demonstrated to vary according to a bee's activity in the colony, they are similar among bees from different castes or sexes. These changes in feeding behavior are related to the bee's function and to less competition for nourishment among individuals of the colony. It is also noted that pollen grains took between 6 and 28 h to pass through the digestive tract. Pollen grains are irregularly accumulated in the various regions of the midgut, which may reflect functional differentiation throughout the midgut. © 2001 Elsevier B.V.

Pollen morphology in Brazilian species of Codonanthe (Mart.) Hanst. and Nematanthus Schrader (Gesneriaceae)

Pollen morphology of 24 Brazilian species of Codonanthe and Nematanthus (Gesneriaceae) was investigated by means of light microscopy, scanning and transmission electron microscopy, in search of new characters that might contribute to taxonomic characterisation of the genera. The pollen grains are tricolpate or tricolporate, semitectate, reticulate, with simplicolumellate muri, sometimes with microreticulum and perforations, or duplicolumellate muri ring-shaped around lumina. The species studied were grouped into five pollen types on the basis of aperture characteristics and details of surface ornamentation. Sub-types were defined with reference to presence or absence of microreticulum and perforations around the lumina. Based on analysis of the morphology and measurements of pollen grains, we verified a close relationship among the genera, but can nevertheless discriminate their species. © 2013 Copyright Collegium Palynologicum Scandinavicum.

Pollen morphology of Fridericia Mart. (Bignoniaceae) from Brazilian forest fragments

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nutritive value and apparent digestibility of bee-collected and bee- stored pollen in the stingless bee, Scaptotrigona postica Latr. (Hymenoptera, Apidae, Meliponini)

In Meliponini bees, pollen is stored inside the hive. We compared the nutritional value and apparent digestibility of bee-collected and bee-stored pollen of Scaptotrigona postica. The results showed no differences in nutritional value and apparent digestibility between bee-collected and bee- stored pollen, suggesting that the storage process of pollen grains do not play a role in these parameters. These results are discussed in relation to other aspects of pollen storage, such as food conservation and/or organoleptic properties of stored pollen.

Pollen morphology of the genus Eremanthus Less. (Vernonieae, Asteraceae)

(Pollen morphology of the genus Eremanthus Less. (Vernonieae, Asteraceae)). In order to evaluate the significance of the pollen morphology for generic and infrageneric taxonomy of the genus Eremanthus (Vernonieae, Asteraceae), and to provide additional data for its phylogenetic reconstruction, the pollen of 20 of the 23 species of the genus was examined using light and scanning electron microscopy. Acetolysed pollen grains were measured, described, and illustrated using light microscopy, while non-acetolysed pollen grains were analyzed using scanning electron microscopy. Pollen grains of these species are isopolar, oblate-spheroidal in most of the species, more rarely prolate spheroidal or suboblate, subtriangular amb, tricolporate and subechinolophate. The variation among quantitative characters does not correlate with the macromorphological subdivision of the genus or with the generic or specific limits.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.