Micro-arrayed wheat seed and grass pollen allergens for component-resolved diagnosis.

BACKGROUND: Wheat is a potent allergen source and can cause baker's asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker's asthma, wheat-induced food allergy and grass pollen allergy. METHODS: We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker's asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts. RESULTS: We identified recombinant wheat flour allergens, which are specifically recognized by patients suffering from baker's asthma, but not from patients with food allergy to wheat or pollen allergy. rPhl p 1 and rPhl p 5 were identified as marker allergens specific for grass pollen allergy. They can be used to replace grass pollen extracts for allergy diagnosis and to identify grass pollen allergic patients among patients suffering from baker's asthma and wheat-induced food allergy. Profilin was identified as a cross-reactive allergen recognized by patients suffering from baker's asthma, food and pollen allergy. CONCLUSIONS: Our results indicate that it will be possible to design serological tests based on micro-arrayed recombinant wheat seed and grass pollen allergens for the discrimination of baker's asthma, wheat-induced food allergy and grass pollen allergy.

Steroid Hormones Stimulate Germination and Tube Growth of in Vitro Matured Tobacco Pollen.

A study of the effects of different steroids on germination and tube growth of tobacco pollen (Nicotiana tabacum L. cv Petit Havana SR1) matured in vitro is presented. Application of the mammalian steroid sex hormones (testosterone, progesterone, and estradiol) resulted in a stimulation of pollen germination and tube elongation. The presence of both steroids and flavonols in the germination medium strongly enhanced the growth of tobacco male gametophytes.

Isolation and characterisation of two wheat beta-expansin genes expressed during male gametophyte development.

Two novel beta-expansin genes, TaEXPB1 and TaEXPB2, were isolated from wheat microspores by suppression subtractive hybridisation. Northern blot and reverse transcription PCR analyses showed that the expression of both genes was restricted to early stages of male gametophyte development (from microspores to immature pollen). A homology search showed high similarity of the newly discovered genes to generative beta-expansins in grass pollen (group 1 pollen allergens). Southern hybridisation revealed that the isolated genes belong to a distinct group within the subfamily of beta-expansin genes in the wheat genome. A comparison of full-length cDNAs with the corresponding genomic sequences showed that there are two introns in the TaEXPB1 gene, whereas TaEXPB2 has three introns. Both genes were predicted to encode highly similar basic proteins (pI 9.0) with molecular masses of approximately 29 kDa consisting of a signal peptide, catalytic, and polysaccharide binding domains, which include conserved cysteines and tryptophans and motifs characteristic for beta-expansins.

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo.

The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type- microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo.

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies.

Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.

A developmentally regulated MAP kinase activated by hydration in tobacco pollen.

A novel mitogen-activated protein (MAP) kinase signaling pathway has been identified in tobacco. This pathway is developmentally regulated during pollen maturation and is activated by hydration during pollen germination. Analysis of different stages of pollen development showed that transcriptional and translational induction of MAP kinase synthesis occurs at the mid-bicellular stage of pollen maturation. However, the MAP kinase is stored in an inactive form in the mature, dry pollen grain. Kinase activation is very rapid after hydration of the dry pollen, peaking at approximately 5 min and decreasing thereafter. Immunoprecipitation of the kinase activity by an anti-phosphotyrosine antibody is consistent with the activation of a MAP kinase. The kinetics of activation suggest that the MAP kinase plays a role in the activation of the pollen grain after hydration rather than in pollen tube growth.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.

Green fluorescent protein (GFP) as a marker during pollen development.

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollen-specific promoters throughout pollen development in Nicotiana tabacum, Arabidopsis thaliana and Antirrhinum majus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each GFP construct was compared in parallel experiments to that of beta-glucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFP-expressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollen-expressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a non-destructive marker for plant reproductive biology and development.

Temporal and tissue-specific expression of the tobacco ntf4 MAP kinase.

The large number of mitogen-activated protein (MAP) kinase genes identified to date in plants suggests that their encoded proteins have a wide array of functions in development and physiological responses, as has been indicated by studies on the factors which lead to the activation of these kinases. Signalling pathways involving members of a multigene family employ a variety of mechanisms to ensure response specificity, one of which is via differential gene expression. We have performed detailed analyses of the expression of the tobacco ntf4 MAP kinase gene using a variety of approaches. The ntf4 gene promoter region was isolated and a chimeric ntf4 promoter-GUS fusion construct was introduced into plants. GUS expression was detected in pollen, in developing and mature embryos, and shortly after seed germination, but not in other floral tissues and tissues such as leaf, root, or stem. This expression pattern was confirmed by northern and western analyses. In situ hybridization and immunolocalization studies showed that the expression of the ntf4 gene and its encoded protein p45Ntf4 occurred in embryos at least from the globular embryo stage until the mature seed, as well as in the seed endosperm. Taken together, the results show that the p45Ntf4 MAP kinase has a very restricted expression pattern, being found only in pollen and seeds. These findings should be important when considering MAP kinase function in plants.

Flavonols stimulate development, germination, and tube growth of tobacco pollen.

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 mum), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.