Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism

Trabajo presentado al Annual Meeting of the American Society of Agronomy and Crop Science Society and Soil Science Society of America, celebrado en Cincinnati (USA) del 21 al 24 de octubre de 2012.Profilin, one of the major allergen (Ole e 2) of olive (Olea europaea L.) pollen, are broadly distributed actin-monomer-binding proteins (ABP). They display a major regulatory role in actin cytoskeleton dynamics, driving cell morphogenesis, sexual reproduction, and translating signals into cellular responses to different environmental stresses. Plants exhibit multiple profilin isoforms w ith distinctive biochemical properties, and differentially regulated. How ever, it is still an open question w hether these profilin isoforms, generated by multiple gene sequence polymorphism, are functionally different, as well as the role of that polymorphism in pollen allergy. Particularly, in differential epitopes generation, profilin isoforms sensitization and cross-reactivity among cultivars, and even among species. In the present study, w e have used mature pollen from olive, birch, hazel, timothy-grass, and maize, in addition to olive germinating pollen and seeds, w ith the aim to analyze the immune-reactivity and subcellular localization of profilin by using polyclonal and specific isoforms antibodies against olive and maize profilins. The results show ed immune-reactivity differences betw een the five species analyzed, betw een olive cultivars, as w ell as between reproductive and vegetative profilins. Furthermore, the existence of different profilin isoforms w as revealed along pollen germination stages. A differential subcellular distribution of profilin isoforms w as found in olive pollen. They w ere localized in the nucleus, pollen aperture regions, pollen and tube w alls and pollen tip, in addition to a general cytoplasmic distribution, in comparison to controls. Data suggest that profilin family might contain numerous functionally distinctive isoforms, spatial-temporal differentially expressed and regulated during vegetative development, pollen maturation and pollen tube grow th. Furthermore, differential immune-reactivity revealed in the study might point out the involvement of common shared and specific epitopes, generated by sequence polymorphism, in differential olive pollen cultivar sensitization of allergenic patients, and cross-reaction to pollen from different species.This study was supported by the following European Regional Development Fund cofinanced grants: MCINN BFU 2004-00601/BFI, BFU 2008-00629, BFU2011-22779, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274, P2011-CVI-7487, P2011-CVI-7487, and by the coordinated project Spain/Germany MEC HA2004-0094.Peer reviewe

Lipid Composition and Associated Gene Expression Patterns during Pollen Germination and Pollen Tube Growth in Olive (Olea europaea L.)

Pollen lipids are essential for sexual reproduction but our current knowledge regarding lipid dynamics in growing pollen tubes is still very scarce. Here, we report unique lipid composition and associated gene expression patterns during olive pollen germination. Up to 376 genes involved in the biosynthesis of all lipid classes, except suberin, cutin and lipopolysaccharides, are expressed in the olive pollen. The fatty acid profile of the olive pollen is markedly different compared with other plant organs. Triacylglycerol, containing mostly C12-C16 saturated fatty acids, constitutes the bulk of olive pollen lipids. These compounds are partially mobilized, and the released fatty acids enter the β-oxidation pathway to yield acetyl-CoA, which is converted into sugars through the glyoxylate cycle in the course of pollen germination. Our data suggest that fatty acids are synthesized de novo and incorporated into glycerolipids by the “eukaryotic pathway” in elongating pollen tubes. Phosphatidic acid is synthesized de novo in the endomembrane system during pollen germination and seems to have a central role in the pollen tube lipid metabolism. The coordinated action of fatty acid desaturases FAD2-3 and FAD3B might explain the increase of linoleic and alpha-linolenic acids observed in the germinating pollen. A continuous synthesis of triacylglycerol by the action of DGAT1 enzyme, but not PDAT, seems also plausible. All these data allow for a better understanding of the lipid metabolism during the olive reproduction process, which can impact in the future in the increase of olive fruit yield and, therefore, olive oil production

Occasional paternal inheritance of the germline-restricted chromosome in songbirds

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby out-competing other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness

Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational, T- and B-Cell Epitopes

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases. [EN]This study was supported by the following European Regional Development Fund co-financed grants: MCINN BFU 2004-00601/BFI, BFU 2008-00629, BFU2011-22779, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274 and P2011-CVI-7487, and by the coordinated project Spain/Germany MEC HA2004-0094. JCJ-L thanks Spanish CSIC and the European Marie Curie research program for his I3P-BPD-CSIC, and PIOF-GA-2011-301550 grants, respectively.Peer reviewe

Characterization of Profilin Polymorphism in Pollen with a Focus on Multifunctionality

Profilin, a multigene family involved in actin dynamics, is a multiple partners-interacting protein, as regard of the presence of at least of three binding domains encompassing actin, phosphoinositide lipids, and poly-L-proline interacting patches. In addition, pollen profilins are important allergens in several species like Olea europaea L. (Ole e 2), Betula pendula (Bet v 2), Phleum pratense (Phl p 12), Zea mays (Zea m 12) and Corylus avellana (Cor a 2). In spite of the biological and clinical importance of these molecules, variability in pollen profilin sequences has been poorly pointed out up until now. In this work, a relatively high number of pollen profilin sequences have been cloned, with the aim of carrying out an extensive characterization of their polymorphism among 24 olive cultivars and the above mentioned plant species. Our results indicate a high level of variability in the sequences analyzed. Quantitative intra-specific/varietal polymorphism was higher in comparison to inter-specific/cultivars comparisons. Multi-optional posttranslational modifications, e.g. phosphorylation sites, physicochemical properties, and partners-interacting functional residues have been shown to be affected by profilin polymorphism. As a result of this variability, profilins yielded a clear taxonomic separation between the five plant species. Profilin family multifunctionality might be inferred by natural variation through profilin isovariants generated among olive germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, affecting the mechanisms underlying the transmission of signals throughout signaling pathways in response to different stress environments. Moreover, elucidating the effect of profilin polymorphism in adaptive responses like actin dynamics, and cellular behavior, represents an exciting research goal for the future

Legumes as food ingredient: characterization, processing, and applications

Editores: Jiménez-López, José Carlos (CSIC); Clemente, Alfonso (CSIC

Whole-Organ analysis of calcium behaviour in the developing pistil of olive (<it>Olea europaea </it>L.) as a tool for the determination of key events in sexual plant reproduction

Abstract Background The pistil is a place where multiple interactions between cells of different types, origin, and function occur. Ca2+ is one of the key signal molecules in plants and animals. Despite the numerous studies on Ca2+ signalling during pollen-pistil interactions, which constitute one of the main topics of plant physiology, studies on Ca2+ dynamics in the pistil during flower formation are scarce. The purpose of this study was to analyze the contents and in situ localization of Ca2+ at the whole-organ level in the pistil of olive during the whole course of flower development. Results The obtained results showed significant changes in Ca2+ levels and distribution during olive pistil development. In the flower buds, the lowest levels of detectable Ca2+ were observed. As flower development proceeded, the Ca2+ amount in the pistil successively increased and reached the highest levels just after anther dehiscence. When the anthers and petals fell down a dramatic but not complete drop in calcium contents occurred in all pistil parts. In situ Ca2+ localization showed a gradual accumulation on the stigma, and further expansion toward the style and the ovary after anther dehiscence. At the post-anthesis phase, the Ca2+ signal on the stigmatic surface decreased, but in the ovary a specific accumulation of calcium was observed only in one of the four ovules. Ultrastructural localization confirmed the presence of Ca2+ in the intracellular matrix and in the exudate secreted by stigmatic papillae. Conclusions This is the first report to analyze calcium in the olive pistil during its development. According to our results in situ calcium localization by Fluo-3 AM injection is an effective tool to follow the pistil maturity degree and the spatial organization of calcium-dependent events of sexual reproduction occurring in developing pistil of angiosperms. The progressive increase of the Ca2+ pool during olive pistil development shown by us reflects the degree of pistil maturity. Ca2+ distribution at flower anthesis reflects the spatio-functional relationship of calcium with pollen-stigma interaction, progamic phase, fertilization and stigma senescence.</p