Release of allergen-bearing cytoplasm from hydrated pollen: a mechanism common to a variety of grass (Poaceae) species revealed by electron microscopy.

BACKGROUND: The release of submicronic particles from grass pollen after rainfall was suggested to be responsible for outbreaks of grass pollen asthma. Recently, we provided evidence for the release of respirable allergen-bearing particles from hydrated ryegrass (Lolium perenne ) pollen as a possible explanation for this phenomenon. OBJECTIVE: We investigated whether water-induced release of respirable allergen-bearing particles could be a mechanism common to several members of the sweet grass family Poaceae (Gramineae). METHODS: Pollens from 6 different Poaceae species were hydrated in water and examined by means of scanning electron microscopy for release of cytoplasmic materials. Rabbit antisera raised against purified recombinant group 1 and 5 allergens were used for immunogold labeling of expelled materials by means of field emission scanning electron microscopy. In addition, group 1 and 5 allergens were immunogold-localized on ultrathin sections. RESULTS: Fresh Poaceae pollens expelled cytoplasmic materials containing group 1 and 5 allergens on hydration in water. Expulsion of submicronic particles strongly decreased after 1 month of storage. CONCLUSIONS: Our results suggest expulsion of cytoplasm after hydration as a mechanism common to pollens of important allergenic grasses. The water-induced release of respirable allergen-bearing particles from grass pollens might explain asthma attacks observed after rainfall during the grass pollen season.

The nature of oxygen in sporopollenin from the pollen of Typha angustifolia L.

Native and peracetylated sporopollenin from the pollen of Typha angustifolia L. was investigated using several spectroscopic methods, inducing Fourier transform infrared spectroscopy (FTIR), solid-state 13C-nuclear magnetic resonance spectroscopy (13C-NMR) and X-ray photoelectron spectrometry (XPS). Interpretation of the experimental data shows that the greater part of oxygen found in sporopollenin originates from hydroxyl groups and must be derived from aliphatics and not from aromatics. This result indicates that not only aromatics and long unbranched aliphatics but also poly-hydroxyl aliphatic components are involved in the complex structure of the polymer. Furthermore, it is most probable that the monomers of the sporopollenin skeleton are linked by ether- and not by ester-linkage. Two possible approaches are suggested for the characterisation of sporopollenin structure.

A new protocol to prepare dry plant specimens for electron microscopy and immunocytochemistry.

Samples from dry kidney bean cotyledons were fixed in acrolein vapor and embedded in Lowicryl K4M using a modified dehydration and embedding technique. The preservation of morphology as investigated by transmission electron microscopy was excellent showing desiccated cells and organelles in life-like preservation. Postembedding immunogold labeling for kidney bean purple acid phosphatase (KbPAP) showed association of kbPAP with ribosome-rich areas of the cytoplasm.

p-Coumaric acid - a monomer in the sporopollenin skeleton.

Sporopollenin obtained from wings of Pinus mugo (Turra) pollen was analysed by pyrolysis mass spectrometry. In the spectrum, mass peaks which are characteristic for p-coumaric acid were dominant. p-Coumaric acid was the main degradation compound when the wing material was treated by a gentle method using AII3, and also when the remaining residue of the treated sporopollenin material was saponified. It is therefore assumed that p-coumaric acid is a genuine structural unit in the sporopollenin skeleton. In addition, the effects of AII3 treatment indicate that the p-coumaric acid might be bound by ether linkages.

Chalcone synthases from spinach (Spinacia oleracea L.) : I. Purification, peptide patterns, and immunological properties of different forms.

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.

Chalcone synthases from spinach (Spinacia oleracea L.) : II. Immunofluorescence and immunogold localization.

The distribution of the two chalcone synthases in leaves ofSpinacia oleracea L. was studied at both the tissue and the subcellular level using immunofluorescence and immunogold techniques. Neither technique differentiated between the two enzyme forms. The chalcone synthases are located in the upper and the lower epidermis and to a minor extent in the subepidermal layers. Traces of the two enzyme forms may be present in the residual mesophyll. This distribution is independent of leaf age. A similar distribution of chalcone synthase among tissues was observed in parsley, pea, and bean. Chalcone synthase is also present in guard cells. The spinach chalcone synthases are cytosolic enzymes, and are not associated with tonoplast or endoplasmic reticulum. A small fraction of the chalcone synthases is located in the stroma of the chloroplasts.

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers.

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip "Apeldoorn" have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.

The systematic distribution of ferulic Acid-sucrose esters in anthers of the liliaceae1.

In an anther survey of 157 di- and monocotyledons, two very rare cinnamic acid conjugates-di- and triferuloylsucrose-were found to occur exclusively in the Liliales and most predominantly in the Liliaceae (Tulipeae, Lilieae, Lloydieae). Whereas the triferuloyl ester was found to be abundant in most of the 20 species and cultivars of TULIPA investigated, only the diferuloyl ester could be detected by the applied methods in FRITILLARIA and LILIUM, in 12 and 9 species and cultivars, respectively. Di- and/or triferuloylsucrose could also be detected in one GAGEA and three ERYTHRONIUM species. Three out of 20 IRIS species investigated, also show the occurrence of these rare esters. This sporadic appearance in IRIS is, at present, considered to be erratic. We suggest that the ferulic acid-sucrose esters might be useful as marker substances for the Liliaceae.

The occurrence of enzymes involved in phenylpropanoid metabolism in the tapetum fraction of anthers.

The contents of another loculus were separated in a pollen and tapetum fraction. The following enzymes involved in phenylpropanoid metabolism were present in the tapetum fraction: shikimate dehydrogenase; phenylalanine ammonialyase; cinnamic acid 4-hydroxylase; SAM (S-adenosylmethionine): caffeate 3-O-methyltransferase; hydroxycinnamate: CoA ligase; "flavanone synthase"; chalcone-flavanone isomerase; SAM: 3',4'-dihydroxyflavonoid 3'-O-methyltransferase; O-glucosyltransferase. It is postulated, that these enzymes derived from the tapetum catalyze the different steps of phenylpropanoid metabolism at or in cavities of the exine after their transfer into the loculus.

[Relations between enzymes which build up flavonols, enzymes which convert flavonols, and the accumulation of phenylpropanoid compounds during the development of anthers]. / über die Beziehungen zwischen flavonolaufbauenden Enzymen, einem flavonolumwandelnden Enzym und der Akkumulation phenylpropanoider Verbindungen während der Antherenentwicklung.

The difference in activity of enzymes which are involved in the biosynthesis of flavonols (phenylalanineammonium-lyase, chalcone-flavanone isomerase) and of an enzyme which converts flavonols is studied during the development of anthers in Tulipa cv. Apeldoorn. The results are considered in relation to the accumulation of simple phenylpropanes, of an intermediate a chalcone and of different flavonoid compounds.In the stages of development with high activities of flavonol synthesizing enzymes there are also high activities in the flavonol converting enzymes. In these stages a large amount of derivates of p-coumaric acid and ferulic acid is accumulated as well as the intermediate product 2',3,4,4',6'-pentahydroxychalcone; accordingly only traces of flavonols can be found.An intensive accumulation of different flavonols does not start before the late phase of development of the anthers in which the stationary concentration of chalcones decreases. It is the stage when the activity of the flavonol converting enzyme decreases rapidly.The relations between the stationary concentration of flavonoid compounds and the interlacing of synthesis and turnover are discussed.

[Chalcone-flavanone isomerase activity and accumulation of phenylpropanoid compounds in anthers]. / Aktivität der Chalkon-Flavanon Isomerase und Akkumulation von phenylpropanoiden Verbindungen in Antheren.

Chalcone-flavanone isomerase from the anthers of Lilium candidum and Tulipa cv. "Apeldoorn" exhibits a distinct substrate specificity. The enzyme catalyses only the isomerization of 2',4,4',6'-tetrahydroxychalcone, whereas it is not active with 2',4,4'-trihydroxychalcone.During the final stage in the development of the anthers, differing isomerase activities were observed. Maximum enzyme activity was measured at a point when the concentration of chalcones was decreasing rapidly and the concentration of flavonols was increasing. These findings strongly support the suggestion that the isomerase plays an important role in flavonoid metabolism.

[Synthesis of phenylpropanes during pollen development]. / Die synthese von phenylpropanen während der Pollenentwicklung.

The synthesis and accumulation of several phenylpropanes in the anther content (pollen+tapetum fraction) during microsporogenesis has been investigated by chromatographic techniques in Narcissus pseudonarcissus, Lilium candidum, and in the Darwin tulip "Apeldoorn".In these species, the pigmentation process is initiated by the synthesis of several cinnamic acid derivates (mainly derivates of ferulic acid) during meiosis II. In Narcissus, and intense synthesis of kaempferol glycosides takes place during the separation of the tetrad which follows immediately upon its formation. In Tulipa and Lilium, however, chalcones are synthesized in an intermediate phase before flavonols and anthocyanins (in Tulipa) are produced in significant amounts.In Tulipa, the investigations revealed the following sequence in the pigmentation process: cinnamic acid derivatives-chalcone-flavonols-anthocyanins. The sequence is discussed in relation to flavonoid biosynthesis. Because of biogenetic considerations a special emphasis is laid on the "chalcone stage". Chromatographic and spectroscopic data show that the isomerization product of the chalcone is eriodictyol. Accordingly, this chalcone must be 2',3,4,4',6'-pentahydroxychalcone. Other chalcones could not be identified.During anthesis the following aglycones are accumulated in the pollen of Tulipa cv. "Apeldoorn": ferulic acid, p-coumaric acid, kaempferol, quercetin, isorhamnetin, delphinidin, and small traces of the pentahydroxy-chalcone, which is the main pigment in the intermediate stages of microsporogenesis.On the basis of histochemical findings, it is suggested that at least the final steps of synthesis leading to flavonol and anthocyanidin glycosides take place on the pollen wall in the loculus of the anthers, that is, in the extracellular space.

[Changes of flavonol and anthocyanin content during microsporogenesis]. / Über die Veränderungen des Flavonol- und Anthocyaningehaltes während der Mikrosporogenese.

The changes in the content of flavonols, anthocyanins, and carotenoids which can be observed in the anther during microsporogenesis were followed in Narcissus pseudonarcissus and in the Darwin tulip "Apeldoorn".The investigations revealed a distinct relationship between the process of pigmentation and the cytologic development in the anther. A marked increase in the production of flavonols occurs during and immediately after the separation of the microspores of the tetrad and seems to be connected in some specific manner with the presence of the immature pollen. In Narcissus, the total flavonol content of the anthers and their pollen and tapetum fraction reaches its highest value during the enclosure of the bud by the bulb and remains more or less unchanged until the flowers open. In contrast, an intense synthesis of flavonols does not begin in the tulip before the flower-bud has left bulb; then the flavonol content increases continuously until anthesis is reached.The colouring of the pollen by anthocyanins does not occur until the final stages of maturation.Without exceptions the production of carotenoids takes place after the separation of the microspores of the tetrad.