A comparative ultrastructural analysis of exine pattern development in wild-typeArabidopsis and a mutant defective in pattern formation

Summary In order to identify factors necessary for the establishment of the reticulate pollen wall pattern, we have characterized a T-DNA tagged mutant ofArabidopsis thaliana that is defective in pattern formation. This study reports the results of an ultrastructural comparison of pollen wall formation in the mutant to wall development in wild-type plants. Pollen wall development in the mutant parallels that of wild-type until the early tetrad stage. At this point in wild-type plants, the microspore plasma membrane assumes a regular pattern of ridges and valleys. Initial sporopollenin deposition occurs on the ridges marking the beginning of probacula formation. In contrast, the plasma membrane in the mutant appears irregular with flattened protuberances and rare invaginations. As a result, the wild-type regular pattern of ridges and valleys is not formed. Sporopollenin is randomly deposited on the plasma membrane and aggregates on the locule wall; it is not anchored to the membrane. Our finding that the mutation blocks the normal invagination of the plasma membrane and disrupts the proper deposition of sporopollenin during wall formation suggests that the mutation could be in a gene responsible for pattern formation. These results also provide direct evidence that the plasma membrane plays a critical role in the establishment of the pollen wall pattern.     

Chemical analysis of osmium tetroxide staining in adipose tissue using imaging ToF-SIMS

Osmium tetroxide (OsO₄) is a commonly used stain for unsaturated lipids in electron and optical microscopy of cells and tissues. In this work, the localization of osmium oxide and specific lipids was independently monitored in mouse adipose tissue by using time-of-flight secondary ion mass spectrometry with Bi cluster primary ions. Substance-specific ion images recorded after OsO₄ staining showed that unsaturated C18 fatty acids were colocalized with osmium oxide, corroborating the view that osmium tetroxide binds to unsaturated lipids. In contrast, saturated fatty acids (C14, C16 and C18) and also unsaturated C16 fatty acids show largely complementary localizations to osmium oxide. Furthermore, the distributions of saturated and unsaturated diglycerides are consistent with the specific binding of osmium oxide to unsaturated C18 fatty acids. The abundance of ions, characteristic of phospholipids and proteins, is strongly decreased as a result of the osmium staining, suggesting that a large fraction of these compounds are removed from the tissue during this step, while ions related to fatty acids, di- and triglycerides remain strong after osmium staining. Ethanol dehydration after osmium staining results in more homogeneous distributions of osmium oxide and unsaturated lipids. This work provides detailed insight into the specific binding of osmium oxide to different lipids.     

Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae)

Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA ethanolic phosphotungstic acid - PA periodic acid - UA/Pb uranyl acetate/lead     

Antibodies to pollen exines

A polyclonal antiserum and monoclonal antibodies have been prepared to purified pollen exines of Calocedrus decurrens Florin. The location of the antigen is in the exine, as shown by light-and electron-microscopic immunocytochemistry. The greatest reduction in antibody binding follows treatment of the exine with chemicals known to alter sporopollenin. These results provide evidence that sporopollenin is antigenic. Exines of ten species of gymnosperms and angiosperms also bound the polyclonal antiserum, indicating similarity of sporopollenin structure.     

Ultrastructure of pollen exine in centrospermous families

Transmission electron microscopy was utilized to examine pollen walls of selected taxa in theAizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Chenopodiaceae, Didiereaceae, Halophytaceae, Nyctaginaceae, Phytolaccaceae, Portulacaceae, andCaryophyllaceae. This conspectus is an adjunct to scanning electron microscope observations of pollen surfaces and is directed towards elucidating the basic wall structure for these families. Although differences in internal morphology were observed at the inter- and intra-familial levels, they were interpreted as reflecting variations rather than major differences. The data indicate close morphological similarities of the first ten families enumerated above, i.e., those containing betalains. TheCaryophyllaceae, an anthocyanin family, indicated a slightly greater heterogeneity of pollen ultrastructure but not to the extent of disassociating it from the betalain families. In fact, this heterogeneity was rivaled by comparable heterogeneity among and within some of the betalain families. The conclusion is that all families have close pollen morphological relationships.Presented in the Symposium Evolution of Centrospermous Families, during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Spatial congruence between exine pattern,microtubules and endomembranes in Vigna pollen

The role of microtubules and endomembranes in pollen wall pattern formation in Vigna vexillata L. was examined using fluorescence laser scanning confocal microscopy. Indirect immunofluorescence using anti--tubulin antibodies revealed that the arrangement of the cortical microtubular cytoskeleton in microspores resembled the reciprocal of the reticulate ektexine ornamentations of mature V. vexillata pollen. Patches of microtubules in cortical cytoplasm corresponded in location with the lumina of the exine reticulum and with apertural sites. Microtubules were absent from cytoplasm under muri (ridges) of the exine reticulum. Labeling of microspores during the mid-tetrad stage with the endomembrane-specific fluorochrome DiOC₆ produced a pattern similar to that of the microtubules; i.e., DiOC₆ staining was localized in cytoplasm underlying lumina and absent from cortical cytoplasm underlying sites of muri. This report represents the first observation of congruence of the pattern of occurrence of any subcellular organelles with exine pattern and, in particular, the congruence of both microtubules and endomembranes in cortical cytoplasm with the lumina of the reticulate exine.     

Immunocytochemical localization of phenolic compounds in pollen walls using antibodies againstp-coumaric acid coupled to bovine serum albumin

Summary Two different antibodies against bovine serum albumin (BSA)-p-coumaric acid-conjugates were produced and used to localize phenolic compounds in exines of pollen from different species,p-Coumaric acid (pC) was coupled to BSA either via the carboxy group (BSA-pC) or directly to the aromatic ring system (BSA-azopC). The polyclonal antibodies raised in rabbits were characterized by ELISA with homologous and heterologous antigens using turkey ovalbumin as carrier protein. The results showed that the two immune sera directed against BSA-pC and BSA-azo-pC, respectively, were specific forp-coumaric acid and structurally similar compounds. Only a very poor binding by acetic acid-ovalbumin-conjugates and no binding by turkey ovalbumin was detectable. The antibodies reacted with partially purified pollen walls and with highly purified exines. The intensity of the immune reaction was proved to be dependent upon the pollen source and the preparation of the pollen walls. Using light and electron microscopy, it was shown for the first time that, in the exines ofCucurbita maxima, antibody binding was predominantly observed in the region of the germ pore apertures, the outer foot layers, and in the micro- and macrospines. We conclude from this and other earlier published data that phenols are important structural compounds of sporopollenin.Abbrevations AA acetic acid - BA benzoic acid - BSA bovine serum albumin - BSA-azo-pC p-coumaric acid coupled in meta position to BSA by a diazo reaction - BSA-azo-pC I immune serum against BSA-azo-pC - BSA-pC p-coumaric acid coupled to BSA via the COOH-group - BSA-pC I immune serum against BSA-pC - FA ferulic acid - OVA ovalbunin from turkey - pC p-coumaric acid - pHY p-hydroxybenzoic acid - SA sinapic acid - SYA syringic acid - TAT TBS-azide-Tween-buffer - TFA trifluoroacetic acid - VA vanillic acid     

The tubular exine ofLauraceae andHernandiaceae,a novel type of exine structure in seed plants

The pollen grains ofLauraceae andHernandiaceae are characteristic in having a tubular exine of ± microfibrillar structure. This is coated with a layer of medium electron-dense globules and very peculiar spines, made up of a substance differing from that of the exine. Such a pollen wall structure is otherwise unknown in seed plants and thus adds to the list of odd features erratically present in various taxa of the Ranalean complex.     

An ultrastructural,cytochemical and immunofluorescence study of postmeiotic development of plasmodial tapetum inTradescantia virginiana L. and its relevance to the pathway of sporopollenin secretion

Summary Establishment of a tapetal plasmodium in postmeiotic stages in anther locules ofTradescantia virginiana encloses the tetrads in membrane-limited compartments. The perispore membrane (PSM), around each tetrad, is derived from composite tapetal cell plasma membranes. The tapetum acquires an abundance of ER and ribosomes and by the late tetrad stage the PSM and its underlying cytoplasm exhibit specialized features, studied here by ZnIO impregnation, osmium maceration, application of indirect immunofluorescence employing antitubulin, conventional thin sectioning and the Thiéry reaction. These features include: labyrinthine convolutions of the PSM resulting from migration of membranous sacs and their partial fusion to the PSM, an intimate relationship of tubular ER with the convoluted PSM, and microtubules underlying the PSM and among the membranous sacs. At the same time membrane-bound granules, comparable to but smaller and simpler than tapetal orbicules of secretory tapeta, form in the convolutions. It is postulated that the ER supplies precursors of sporopollenincontaining parts of the spore wall, that the PSM-associated microtubules stabilise the whole secretory apparatus at the tapetum-spore interface, and that the precursors are expelled into the lumen bounded by the PSM and then accreted upon the orbicule-like granules or the developing spore wall. With dissolution of the callosic wall, the plasmodium invades the intermicrosporal spaces of late tetrads, the PSM unfolding its elaborations and becoming closely appressed to the exinous surfaces of individual spores. Microtubules, although present during this phase of invasion, do not seem to propel the invasion processes and may have roles in shape maintenance. During pollen mitosis and enlargement the tapetal cytoplasm accumulates lipidic globules. A late phase of Golgi activity precedes accumulation of vesicles or vacuoles near the spores, these being bounded by single or multiple tripartite membranes. With anther desiccation, portions of plasmodium are deposited on the pollen surface in the form of tryphine, the deposits containing stacked membrane-like bilayers.     

Exine formation in the pollinium ofDendrobium

Summary The position of the callose wall is related to the position of the primexine matrix that forms around the peripheral tetrads during microspore development of the compound unit, the pollinium. We report a combined freeze-fracture and freeze-substitution study of the events associated with early exine development. Stage one of exine development is deposition of protosporopollenin that is probably synthesised by the microspore and secreted to the primexine matrix where it is polymerised. Enzymes for the polymerisation of the protosporopollenin may be synthesised by the microspores and then transported, via the endoplasmic reticulum, to the plasma membrane. Stage two of exine development follows callose dissolution and deposition of tapetally derived sporopollenin. Hence exine form and exine deposition inDendrobium appear to be the result of intimate cooperation between the microspore, the plasma membrane, the callose and the tapetum.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Immunodetection of pectin and arabinogalactan protein epitopes during pollen exine formation of Beta vulgaris L.

Summary. We present the results of ultrastructural and immunocytochemical studies of sugar beet microsporocytes during the developmental phase that begins with the first meiotic metaphase and ends with the formation of young tetrads. The most prominent feature noted during this period of microsporogenesis was the presence of numerous cisternae of endoplasmic reticulum which frequently lie perpendicular to the surface of the plasma membrane and eventually fuse to it. Microscopic observations have been combined with the detection of several carbohydrate epitopes representing pectins and arabinogalactan proteins in the primexine and incipient exine. Pectin domains that possess both low and highly methylesterified epitopes, as well as pectin side chains enriched in (1→4)-β-D-galactose residues, are deposited in this young microspore wall. The epitopes of arabinogalactan protein that bind to JIM13, JIM8, and LM2 antibodies are localised within the callose wall surrounding posttelophase tetrads. The possibility of endoplasmic-reticulum involvement in the synthesis, transport, or metabolism of several microspore wall compounds is discussed. Correspondence and reprints: Institute of Plant Breeding and Acclimatization, Powstańców Wielkopolskich 10, 85-090 Bydgoszcz, Poland.     

Pollen development in a submerged plant,Ottelia alismoides (L.) Pers. (Hydrocharitaceae)

Exine structure and its developmental program in a submerged plant,Ottella alismoides (L.) Per. were investigated with scanning and transmission electron microscopies. Verrucate protrusions initiate on microspore plasma membrane at early tetrad stage. The verrucate protrusions develop into spines during free microspore stage. A foot layer is formed by accumulation of lamellated structure. The pollen grains ofOttelia alismoides are inaperturate, not omniaperturate, because of the well-developed foot layer. The inaperturate pollen grains ofOttelia are characterized by the spinous protrusions and the granular foot layer.     

Pollen morphology ofPyrola and its taxonomic significance

Pollen from 152 collections representing 16Pyrola species was examined with LM, SEM and TEM. The genusPyrola is stenopalynous in having tetrads and 3-colporoidate grains. However, a continuous and serial variation in the exine sculpture and the tetrad diameter (D) was revealed within the genus. Křísa's system ofPyrola (1971) was re-examined in the light of these palynological characters which may have a taxonomic significance, as well as the external morphology. P. minor (only member of the subgenusAmelia), which is characterized by having small (D=30–35 μm) pollen tetrads with verrucate sculpture must best be placed near sectionPyrola of the subgenusPyrola. P. faurieana must be segregated from sectionChlorantha and together withP. media placed intermediate between the subgenusAmelia and sectionPyrola of the subgenusPyrola based on their larger (D=ca. 40 μm) pollen tetrads with verrucate sculpture. Two series of sectionChlorantha excludingP. faurieana are differentiated in the exine sculpture. Therefore, they are regarded as the distinct taxonomic groups. SectionScotophylla is characterized by having psilate sculptured pollen tetrads, which indicates that its sectional rank is sufficiently asserted. The evolutionary trend in the exine sculpture from verrucate through rugulate to psilate is suggested withinPyrola.     

Ultrastructural aspects of storage lipid mobilization in the tapetum of Lilium hybrida var. enchantment

Stages in the formation and degradation of pollenkitt in the anther of Lilium have been investigated using the electron microscope. This material, which appears to be a complex of lipid and carotenoids, is formed during the autolysis of the tapetal cells by the fusion of lipidic inclusions with globules derived from plastids. Autolysis of the tapetal cells is progressive for it commences with the disintegration of many cytoplasmic components, followed by the breakdown of storage lipids. The plasma membrane maintains its integrity during these events apparently, by proliferation, aiding in the transfer of the products of hydrolysis into the loculus. During the course of lipid breakdown, a striking vacuolar system is formed in the tapetal cytoplasm, presumably containing the products of this hydrolysis. The source of membranes for this system is clearly the lipid globules themselves. The generation of the membrane apparently involves the participation of electronopaque material, possibly enzymic, contained within the lipid globules.     

Dimorphic exine sculpturing in three distylous species ofLinum (Linaceae)

DistylousLinum grandiflorum, L. mucronatum, andL. pubescens have dimorphic pollen grains. In short-styled flowers the exine has monomorphic processes each with a ring of papillae. In long-styled flowers the exine has two types of processes: small processes terminating in a spinule and larger processes with a central spinule surrounded by a ring of spinuels, papillae or buttresses. The distylousLinum species of four different sections, in which morph-specific differences in the wall structure of stigmatic papillae have been reported, also exhibit exine dimorphism. The patterns of these dimorphisms are similar in all four sections in which distyly is present.     

Development of the echinate pollen wall inFarfugium japonicum (Compositae: Senecioneae)

Development of the echinate pollen grains inFarfugium (Compositae: Senecioneae) has been studied by a combination of transmission electron microscopy and field emission scanning electron microscopy with a freeze fractured method. The inner surface of the callose wall surrounding each microspore does not possess an echinate pattern before primexine deposition begins. The primexine formation coincides with the initiation of spines. The freeze fractured primexine shows probacula which form transverse rods. The developing exine has an inner spongy substructure. The endexine is formed by the accumulation of the electron dense lamellae with white lines after the dissolution of the callose wall. In the present study, it is confirmed that the developmental process of pollen formation revealed in the field emission scanning electron microscope is consistent with the results obtained using the transmission electron microscope.     

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.Abbreviations HPLC high-performance liquid chromatography - MS mass spectrometry - TLC thin-layer chromatography