ISOLATION OF EXINES FROM GYMNOSPERM POLLEN

Exines of certain Gymnosperms spontaneously separate from the intine during the process of hydration preceding pollen germination. Exines of pollen of Calocedrus decurrens, Chamaecyparis lawsoniana, Juniperus occidentalis, Sequoia sempervirens, and Pseudotsuga menziesii were isolated from hydrated, autoclaved pollen. Free exines were purified by centrifugation on a discontinuous sucrose gradient of densities 1.14 to 1.27 g/ml. The outer intine dissolved on autoclaving. This method may be applicable to a wide range of genera. Purified exines are of potential use in chemical analyses of sporopollenin and in production of antibodies to exine.     

Investigations cytochimiques,marquage immunocytochimique et effets destructeurs des U. V. sur le pollen de Dactylis glomerata L

The elucidation of the ultrastructural cytochemistry, coupled with chemical stabilizing procedures during fixation and embedding, make a significant contribution to the understanding of pollen-exine ontogenesis. A combination of the Con-A agglutination and lanthanum precipitation methods proved particular advantageous during the intine formation stage. By using immunogold techniques, it is possible to demonstrate that only the mature exine of the atmospheric pollen grains reacted positively after sectioning, not the intine. An important difference appears when immature pollen grains are treated under the same conditions: in this case, both the microsporal cytoplasm of the future pollen grain and the immature exine react, but not the intine.

In untreated pollen grains, the exine is a biological material of exceptionally high density with a very low stainability: staining for proteins and lipids is only moderate. A degradation of exine structures by U. V. radiation of 254 nm can be easily proved and the framework obtained is comparable to that induced by some chemical attacks. When sporopollenin is degraded from filamentous sub-units of the exine, stainability increases, and the cytochemical tests for acid-mucopolysaccharids are positive. It is clear that glycocalyx units within exines are chemically bound to the sporopollenin matrix. Attention can also be profitably directed to the future investigation of the function of exine frameworks, through allergen fixation in living pollen grains.     

A comparative study of the gradual degradation of exines,resulting from the effects of temperature

The gradual degradation of three types of pollen exines from different plant groups (gymnosperms and angiosperms) with rising temperature has been observed and comparisons made. Pollen grains are heated to different temperatures (100°C–350°C) in a sealed copper tube, placed in a nichrome wire resistance furnace. In each case the pollen grains are heated for 100 hours.The colour change and the size reduction with rising temperature are common to all pollen types. The sexine or ornamented part of the pollen exine is affected first by rising temperature. In angiosperm pollen, the sexibe pattern is not recognisable at 300°C, but pine pollen retains its pattern up to 350°C. The nexine seems to be more stable at high temperatures than the sexinous elements and either remains unaltered with remnants of the sexinous pattern, or becomes altered and amorphous.The lamellar part of the nexine appears to be important and the evolutionary significance of the exine is discussed. The present work shows that the gymnosperm pollen has more stable exines, and may be better adapted for survival than angiosperm exines.     

5种裸子植物花粉外壁成分及结构分析比较

利用傅里叶变换红外光谱仪对银杏、日本冷杉等5种裸子植物的花粉外壁成分进行测定分析,结果表明,这5种裸子植物花粉外壁的红外光谱主要由蛋白质、脂类及多糖类物质的特征吸收峰组成。但不同科属间的花粉外壁主要成分含量存在较大差异,其中银杏科的银杏花粉外壁以蛋白质含量最为丰富;松科的雪松花粉外壁以脂类物质较为丰富;杉科的日本冷杉、杉木和日本柳杉花粉外壁成分以多糖类物质为主,但种间花粉外壁成分仍存在差异。采用扫描电镜观察5种裸子植物花粉,显示银杏、柳杉和杉木花粉粒体积较小,不具气囊,银杏花粉粒外壁表面具较均一条纹状纹饰,日本柳杉和杉木花粉粒外壁具颗粒状突起。雪松和日本冷杉花粉粒具气囊,体积较大,花粉外壁分别是粗糙具小穴状纹理以及表面光滑具微穿孔。     

Re-Exposure of Tapes at High Temperature and Pressure in the Lycopodium Clavatum Spore Exine

Lamellations are visualizable through the staining commonly used in transmission electron microscopy during exine formation on Lycopodium and other spores, and the nexine of pollen grains. The lamellations so exposed consist-of dark tapes at either side of an unstained (white) line. Neither tapes nor white lines are visualizable in the exine of mature spores of Lycopodium. The continued presence of lamellations having tape-white line spacing has been demonstrated with inorganic tracers in the nexine of pollen in which lamellations otherwise appeared to be absent. Through high contrast staining methods for TEM we have observed lamellations in the residual exine following heat treatment (350[ddot]C) of mature spores of Lycopodium clavatum. The surface of these residual exines was etched by treatment with hot 2-aminoethanol and filaments were observed to protrude from the etched surfaces. The residual exine stained darkly. Lycopodium spores heated to 300[ddot]C at 1 kb pressure had long filaments exposed at the surface of the residual exine (sporopollenin). Sections of the pellet remaining after heat and pressure treatment also included bundles of closely parallel filaments and masses of isolated filaments. The filaments were stained while the exine residue, assumed to include sporopollenin, was not. Isolated filaments produce a stable metachromasia with toluidine blue indicating the presence of many closely spaced sites of negative charge. The staining of intact exines with basic dyes may result from anionic sites on filaments embedded within the exine rather than being due to sporopollenin. The results of our experiments indicate that the filaments are more resistant to heat and pressure and 2-aminoethanol than is sporopollenin. We propose that the trilamellar elements commonly called tapes and white lines might be composed of two filaments bridged by polybasic molecules.     

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.     

Developmental accumulation of hydroxyproline and hydroxyproline-containing proteins in Zea mays pollen

The hydroxyproline content of developing Zea mays (maize) pollen was determined. The level of hydroxyproline in uninucleate microspores early in pollen development was low (0.004% of dry weight). In contrast, mature pollen is enriched for this amino acid (0.1% of dry weight). In mature pollen, 90% of the hydroxyproline is in the soluble fraction. Upon in vitro pollen germination, hydroxyproline associated with the insoluble fraction increased from 10% to 26% of the total hydroxyproline. Antibodies specific to extensins and arabinogalactan proteins (AGPs), two major classes of hydroxyproline-containing proteins, recognized two distinct groups of proteins in maize pollen by Western analysis. The two types of pollen hydroxyproline-containing proteins could also be distinguished based on their behavior upon anion exchange chromatography.     

Taxonomic,evolutionary, and functional considerations ofCompositae pollen ultrastructure and sculpture

Two basic patterns of exine ultrastructure are found in theCompositae, the caveate Helianthoid pattern and the non-caveate Anthemoid pattern. TheHeliantheae, Astereae, Inuleae, Sececioneae, Calenduleae andEupatorieae all have pollen with caveate exines. TheMutisiseae, Vernonieae andCardueae have predominately Anthemoid pollen. TheAnthemideae, Arctoteae andLactuceae have pollen with exines of both patterns. Recent investigations of pollen in theVernonieae suggest that these exine ultrastructures in the family have evolved in response to mechanical stresses on the wall which are caused by changes in volume of the grain as it loses or gains water from its environment.     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.     

Pollen-wall Proteins: 'Gametophytic' and 'Sporophytic' Fractions in the Pollen Walls of the Malvaceae

Proteins are stored in two sites in the pollen grain walls ofthe Malvaceae, (a) in the cellulosic intine, mainly in the vicinityof the circular apertures, and (b) in cavities in the sculpturedlayer of the exine. The intine-held materials are incorporatedduring the growth of the wall. The exine materials are derivedfrom the tapetum, which during dissolution releases cistemaewith a granular-fibrillar content bounded by ribosomal membranes.This fraction is injected into the exine cavities after thecompletion of wall growth through micropores in the tectum.PAS-reacting material is associated with the injected protein.Another tapetal fraction, lipid in nature and commonly containingcarotenoids, remains on the surface of the pollen grains toform the Pollenkitt. While protein can be detected cytochemically in both intineand exine sites, acid phosphatase and ribonuclease activitywas found to be associated only with the former. Immunofluorescencemethods using antiserum to total pollen leachates showed thatantigens are present in both sites. When the pollen grains are moistened, the exine-held proteinsbegin to pass out through the micro-pores in the tectum within30 s of moistening, while the main discharge from the aperturalintine follows in 4–5 min. These observations, together with evidence from other families,suggest that the intine-held proteins of angiosperm pollen grainsare always produced by the male gametophyte, while those heldin exine cavities are sporophytic in origin, being derived fromthe tapetum. As previously proposed, it seems probable thatin intraspecific incompatibility systems of the gametophytictype control is mediated through intine-held ‘recognitionsubstances’, whereas in sporophytic systems the exine-heldmaterials are concerned.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Chemical composition of Azotobacter vinelandii cysts

Cysts of Azotobacter vinelandii ATCC 12837 were germinated by exposure to 3.0 mm ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane buffer at pH 7.8, and their outer coats (exines) were purified by differential and isopycnic centrifugation. Electron micrographs of exine showed it to consist of multilayers of a three-membered sheet structure whose thickness was 7.0 to 7.5 nm. The inner, less electron-dense layer (intine) was also prepared from cysts by EDTA treatment, centrifugation, concentration, and dialysis. The exine consisted of 32% carbohydrate, 28% protein, 30% lipid, and 3.2% ash, with the ash comprised of 1.62% calcium, 0.02% magnesium, and 0.34% phosphorus. The amino acid composition of exine was similar to that of gram-negative bacterial cell walls. The intine consisted of 44% carbohydrate, 9.1% protein, 37% lipid, and 4.1% ash, with the ash comprised of 2.45% calcium, 0.02% magnesium, and 0.38% phosphorus. The carbohydrates of both exine and intine contained glucose, mannose, xylose, and rhamnose. Glucosamine and galactosamine were found only in the exines. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids with 10 to 18 carbon atoms and mono-unsaturated C(11), C(16), and C(18) fatty acids. The exines contained mostly bound lipid, but intines contained primarily free lipid.     

Pollen Morphology of Loxostemon (Cruciferae) in China

The present paper deals with the pollen morphology of 10 species and 1 variety of Loxostemon in China. The pollen grains were all examined under light microscope. The pollen grains of Loxostemon are subspheroidal, spheroidal or prolate, 18--33×11.8-28 μ in size, 3-colpate, colpi 15-21 μ long and 1-2 μ wide. The exine is 1.5-3 μ thick with two indistinct or distinct layers. All the pollen grains are generally reticulate under light microscope. They are distinctly or obscurely and finely reticulate. L. axillus and L. repens are generally similar in gross morphology, but the pollen grains of these two species are different. The pollen grains of L. axillus are regularly polygonally reticulate, colpi are acute-ended and the exine is about 3 μ thick, whereas those of L.repens are irregularly polygonally reticulate, colpi are enlarged at both ends and the exine is about 2.8 μ thick. L. incanus and L. stenolobus appear to have similar gross morphology, but the pollen grains of the former have exines with two distinct layers and a densely and finely reticulate ornamentation and those of the latter have exines with two indistinct layers and a flexuosely reticulate ornamentation.     

SOLUBILITY OF POLLEN EXINES

The sporopollenin of pollen exines of Ambrosia trifida is soluble in fused potassium hydroxide, in strong oxidizing solutions, and in certain organic bases. It is insoluble in other organic and inorganic acids and bases, in lipid solvents, and in detergents. The outer exine layer of gymnosperm and angiosperm pollen dissolves in 2-aminoethanol. The inner exine layer, as well as the exine of pteridophyte spores, is insoluble. The exine dissolution process in 2-aminoethanol involves swelling and disintegration of exine structures, leaving some residual globules. Sporopollenin shares some solubility properties with lignin and cutin but appears to be chemically distinct from these substances.     

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum‐specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate‐shaped compared with the three‐dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild‐type. The wild‐type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2‐Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co‐localized with the endoplasmic reticulum (ER) signal. RNA‐Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.     

Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride

The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion.     

Developmental staging of maize microspores reveals a transition in developing microspore proteins

A method for the preparation of developmentally staged microspores and young pollen from maize (Zea mays) has been devised. The preparations are of sufficient purity and quantity for biochemical analysis, including the analysis of steady-state protein and RNA populations associated with each stage. A major transition in protein populations occurs during the developmental period that encompasses microspore mitosis, the asymmetric nuclear division producing the vegetative and generative nuclei. Several differences between early and late stage proteins can be detected by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional gel electrophoresis of proteins reveals that over half of the steady-state proteins differ between the younger and older stages, either quantitative or qualitative. One protein that increases in relative abundance about fourfold is actin. In vitro translation of RNA isolated from staged microspores demonstrates changes in microspore gene expression during the same developmental period.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

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 Apertural Wall in Pollen of Linum grandiflorum

The apertural wall in tricolpate pollen of Linum grandiflorumwas investigated in order to understand its functioning duringdesiccation and rchydration. Whole and sectioned pollen grainswere studied with light or electron microscopy and by cytochemicalmeans. The areas of the apertures were examined in fresh drypollen, in grains moistened on agar gel or removed from compatiblestigmas, and in pollen from mature undehisced anthers The intine was found to consist of an inner ß-glucanlayer and an outer pectic layer. At the apertures the pecticlayer is thickened and overlaid by a ß-glucan layer.The pectinaceous intine stains red with basic fuchsin. The presenceof a third wall layer, the medine, was not confirmed. The aperturalintine thickenings possess considerable imbibitional capacityand at rehydration they appear as swollen lenticular bodies A procedure is described for obtaining intact exine free grains(EFG's) and whole, separated exines of L. grandiflorum. Invariably,the released EFG's consisted of protoplasts encased in the cellulosicintine. In most grains the outer intine remained attached tothe separated exine In L. grandiflorum the outer wall of the aperture expands whilethe protoplast and endintine are still infolded. Apparently,the exintine becomes detached from the endintine during desiccationand re-attaches at rehydration. It is suggested that the transientdetachment controls the influx of water into the vegetativecell Except for morph-specific exine processes no differences instructure of the aperture wall or its functioning at rehydrationwere observed between pin and thrum grains Pollen wallM, apertures, exintine, exine free grains, rehydration, desiccation, Linum grandiflorum