Analysis of Sporopollenin Isolated from Pollen of Ambrosia Artemisiifolia and A. Trifida

Sporopollenin was isolated from pollen of Lilium longiflorum, Ambrosia trifida and A. artemisiifolia.The sporopollenin was characterized with respect to its infrared spectrum, elemental composition and nature of potassium hydroxide fusion products. Sporopollenin from the Ambrosia species seemed identical. There were only slight differences between Lilium and Ambrosia sporopollenin.     

孢粉素的物理化学性质和生物医学应用研究进展*

孢粉素是类聚乙烯醇链通过酯键和缩醛高度交联的天然生物高分子,构成花粉和孢子的外壁,能够抵抗物理、化学、生物腐蚀,堪称自然界最坚固的有机物,被誉为植物界的金刚石。孢粉素微囊(SEC)自然界来源丰富、生物相容性好、无免疫原性,表面含有丰富的羧基、羟基和酚基,能够功能化或者与其他纳米材料构建复合材料;其表面丰富的纳米孔道增加了材料的比表面积,有利于捕获癌细胞或目标生物分子。SEC独特的性质使其在药物载体、口服疫苗载体、影像诊断、生物传感、细胞生长支架、微反应器、微型机器人等方面得到广泛的应用。阐述了孢粉素的结构、物理化学性质、制备方法和功能化方面的研究进展, 探讨了孢粉素的应用前景、存在的问题以及未来的发展方向。     

Pollen Wall Ontogeny of Felicia muricata (Asteraceae: Astereae)

Pollen wall development in Felicia muricata Thunb. Nees (Asteraceae)was investigated by electron microscopy after addition of thelypophilic dye malachite green to the primary fixative. Pollenwall formation commences with the deposition of fibrillar electrondense units which represent future interbacular spaces. Radiallyarranged trilammelate structures develop between the fibrillarunits. Sporopollenin is deposited in the trilammelate structures.After release of the microspores from the tetrad, formationof interbacular spaces is accompanied by disintegration of fibrillarunits. Endexine formation occurs on white lines of unit membranedimensions. Intine formation follows the pattern described formost angiosperms. Observation of bead-like globules and pollenkittwith a membraneous structure is presumed to be related to theaddition of malachite green to the fixative.Copyright 1993,1999 Academic Press Felicia muricata (Thunb.) Nees, pollen, pollenkitt, ultrastructure, fixation     

High resolution solid state C NMR spectroscopy of sporopollenins from different plant taxa

The extremely chemically resistant component of the cell wall of spores, pollens, and some microorganisms, sporopollenin, is generally accepted to be derived from carotenoids or carotenoid esters. However, we report here that ¹³C NMR analyses of sporopollenin from several sources shows that this widely held view is incorrect, with one possible exception. Sporopollenin is not a unique substance but rather a series of related biopolymers derived from largely saturated precursors such as fatty acids. The biopolymers contain widely varying amounts of oxygen in the form of ether, hydroxyl, carboxylic acid, ester, and ketone groups.     

Continuous decomposition of sporopollenin from pollen of Typha angustifolia L. by acidic methanolysis

Sporopollenin from the pollen of Typha angustifolia L. was exposed to a series of 36 subsequent acidic methanolysis procedures. The remaining decomposition products were investigated using several spectroscopic methods including Fourier transform infrared spectroscopy (FT-IR), solid state 13C nuclear magnetic resonance spectroscopy (13C-CPMAS-NMR) and X-ray photoelectron spectrometry (XPS). Substantial weight losses of the sporopollenin material occur after each acidic methanolysis step, while FT-IR and 13C-CPMAS-NMR spectra display no noticeable differences after 12, 24 and 36 steps. These findings are interpreted as a hint that the sporopollenin polymer has a uniform composition, i.e. relatively small monomer moieties of similar primary structure are present. Moreover, the weight losses account for the presence of substantial amounts of ether linkages in the sporopollenin polymer.     

Ultrastructural characterization of exine development of the transient defective exine 1 mutant suggests the existence of a factor involved in constructing reticulate exine architecture from sporopollenin aggregates

A male-sterile mutant of Arabidopsis thaliana, in which filament elongation was defective although pollen fertility was normal, was isolated by means of T-DNA tagging. Transmission electron microscopy (TEM) analysis revealed that primexine synthesis and probacula formation, which are thought to be the initial steps of exine formation, were defective, and that globular sporopollenin aggregation was randomly deposited onto the microspore at the early uninucleate microspore stage. Sporopollenin aggregation, which failed to anchor to the microspore plasma membrane, was deposited on the locule wall and in the locule at the uninucleate microspore stage. However, visually normal exine with a basic reticulate structure was observed at the middle uninucleate microspore stage, indicating that the exine formation was restored in the mutant. Thus, the mutant was designated transient defective exine 1 (tde1). These results indicated that tde1 mutation affects the initial process of the exine formation, but does not impair any critical processes. Our results also suggest the existence of a certain factor responsible for exine patterning in A. thaliana. The TDE1 gene was found to be identical to the DE-ETIOLATED 2 gene known to be involved in brassinosteroid (BR) biosynthesis, and the tde1 probacula-defective phenotypes were recovered in the presence of BR application. These results suggest that BRs control the rate or efficiency of initial process of exine pattern formation.     

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.     

PpASCL, a moss ortholog of anther-specific chalcone synthase-like enzymes, is a hydroxyalkylpyrone synthase involved in an evolutionarily conserved sporopollenin biosynthesis pathway

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes. We analyzed available P.patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P.patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity. Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl α-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL. These results suggest that ASCL functions together with other sporophyte-specific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.     

FINE STRUCTURAL STUDIES OF ZEA MAYS POLLEN I: CELL MEMBRANES AND EXINE ONTOGENY

Electron microscopy was used to study pollen wall ontogeny in Zea mays. The initial stage of development consisted of compartmentalization of microspores within callose special walls. Microspore plasma membranes retracted and tubular elements of the endoplasmic reticulum became perpendicularly oriented to the plasma membranes. Evaginations of the endoplasmic reticulum into the microspore plasma membrane resulted in the establishment of a template or blueprint of the mature pollen wall. Sporopollenin deposition upon the template began immediately after dissolution of the callose special walls and release of the microspores into the anther locule. The columellae were the first pollen wall units to be formed; the tectum and foot layer became established shortly thereafter. The granular endexine was the last-formed unit. The relationships of membrane systems to the ontogeny of the pollen wall units and the mode of pollen wall growth are discussed.     

Basis for the Resistance of Several Algae to Microbial Decomposition

The basis for the resistance of certain algae to microbial decomposition in natural waters was investigated using Pediastrum duplex, Staurastrum sp., and Fischerella muscicola as test organisms. Enzyme preparations previously found to convert susceptible algae into spheroplasts had no such effect on the resistant species, although glucose and galacturonic acid were released from P. duplex walls. Little protein or lipid but considerable carbohydrate was found in the walls of the refractory organisms, but resistance was not correlated with the presence of a unique sugar monomer. A substance present in Staurastrum sp. walls was characterized as lignin or lignin-like on the basis of its extraction characteristics, infrared spectrum, pyrolysis pattern, and content of an aromatic building block. Sporopollenin was found in P. duplex, and cellulose in Staurastrum sp. Cell walls of the algae were fractionated, and the fractions least susceptible to microbial degradation were the sporopollenin of P. duplex, the polyaromatic component of Staurastrum sp., and two F. muscicola fractions containing several sugar monomers. The sporopollenin content of P. duplex, the content of lignin or a related constituent of Staurastrum sp., and the resistance of the algae to microbial attack increased with age. It is suggested that resistance results from the presence of sporopollenin in P. duplex, a lignin-like material in Staurastrum sp., and possibly heteropolysaccharides in F. muscicola.     

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     

Gas chromatography-mass spectrometric analysis of oxidative degradation products of sporopollenin inMagnolia grandiflora (Magnoliaceae) andHibiscus syriacus (Malvaceae)

Sporopollenin can be dissolved in water by oxidative degradation with using potassium permanganate (KMnO₄). in mature pollen grain ofMagnolia grandiflora andHibiscus syriacus some organic compounds were extracted from the aqueous phase into hexane, and analyzed by using gas chromatography-mass spectrometry (GC-MS). The analysis shows that the extracted compounds Include some organosilicon compounds. By researching of mass spectra data base, the organosillicon compounds are suggested as 1, 1, 1, 5, 7, 7, 7-heptamethyl-3,3-bis(trimethylsilox))tetrasiloxane and 1, 1, 1, 3, 5, 7, 7, 7-octamethyl-3, 3- bis(trimethylsiloxy)tetrasiloxane. 1,2-benzendicarboxylic acid butyl 2-ethylhexyl ester is also suggested by data base research in the extracted compounds. The present study implies that silicon (Si) is considered to be located in inner part of three dimensional structure of sporopollenin.     

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.     

Carbon isotopic composition of Ambrosia and Artemisia pollen: assessment of a C(3) -plant paleophysiological indicator

? There is limited evidence on how shifts in plant physiological performance influence vegetation variations in the paleorecord. ? To evaluate δ(13) C of pollen from C(3) plants as an indicator of community-level physiology, small quantities (10-30 grains) of untreated pollen and sporopollenin from herbarium specimens of Ambrosia (A.?tomentosa and A.?psilostachya) and Artemisia (A.?frigida, A.?ludoviciana and A.?dracunculus), genera abundant in grassland pollen profiles, were isolated by micromanipulation. Their δ(13) C values were measured using a spooling-wire microcombustion device interfaced with an isotope-ratio mass spectrometer. Leaf δ(13) C was also measured. Carbon isotope discrimination (Δ) for untreated pollen, sporopollenin and leaves was compared with historic records of seasonal precipitation amount, vapor pressure deficit and the Palmer Drought Severity Index (PDSI). ? Each species showed positive correlations between Δ of untreated pollen and sporopollenin. Sporopollenin Δ was most strongly correlated with PDSI. Correlations among leaf Δ and moisture indicators were stronger for Ambrosia than Artemisia. ? These results suggest that sporopollenin Δ indicates the level of moisture stress in C(3) plants. Therefore, δ(13) C analysis of pollen promises to help address important paleoecological questions, such as how community-level physiology contributes to shifts in vegetation composition.     

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.     

THE GLYCOCALYX AND INITIATION OF EXINE SPINULES ON MICROSPORES OF CANNA

Spinules of Carina generalis pollen are initiated within a tridimensional network during the microspore tetrad period. The network is stained selectively with the hydrazide-silver proteinate method of Thiéry following periodate oxidation and by phosphotungstic acid at low pH, demonstrating the presence of polyanions. Protein is indicated as a component of the network by positive staining with PTA in acetone. These results suggest the presence of polysaccharides and proteins, possibly as mucopolysaccharides or glycoproteins. The network may be considered as a part of the glycocalyx because of its composition and association with the plasma membrane. Sporopollenin accumulates on the tridimensional network or in meshes of the net. The beaded fine structure of spinules resists the acetolysis mixture of Erdtman. Our results imply that the plasma membrane and its glycocalyx are part of the system which mediates genetic expression of exine form. The implication is compatible with formation of specific exines on all pollen grains of a plant and on aborted microspores, diminutive spores with depauperate chromosome complements, and enucleate bodies of cytoplasm resulting from meiotic abnormalities.     

Enantioselective hydrolysis of rasemic naproxen methyl ester with sol–gel encapsulated lipase in the presence of sporopollenin

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, the Candida rugosa lipase (CRL) was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) in the presence and absence of sporopollenin and activated sporopollenin as additive. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of rasemic Naproxen methyl ester that was studied in aqueous buffer solution/isooctane reaction system. The results indicated that the sporopollenin based encapsulated lipase particularly had higher conversion and enantioselectivity compared to the sol–gel free lipase. In this study, excellent enantioselectivity (E > 400) has been noticed for most lipase preparations (E = 166 for the free enzyme) with an ee value ～98% for S-Naproxen. Moreover, (S)-Naproxen was recovered from the reaction mixture with 98% optical purity.     

Receptor-Independent Sporopollenin

Pollen of some species of the genus Quercus shows rod-shaped substructures in fresh or acetolysed exines, while in other species rod substructure is mostly masked by sporopollenin. Oxidation with potassium permanganate removes exine substance (sporopollenin) from between the rod substructures. We propose that the rods include receptors for sporopollenin. The sporopollenin between rods we refer to as ‘receptor-independent sporopollenin’. Pollen of Typha, when mature, has tectal surfaces with concave tops and sides, whereas during development the tectal surfaces are smoothly rounded. After acetolysis treatment followed by potassium permanganate the tectum surfaces again appear rounded. When these exines are subsequently eroded by a fast atom source, rod-shaped substructures are seen to protrude from the tectum. These structures are equivalent in size and shape to the rods of the exine of Quercus. Sporopollenin that accumulates over and masks rod substrucutre is less resistant to our degradative methods than the sporopollenin in rod structures of exines. We suggest that the exine material we call “receptor-independent sporopollenin” be given a simple positive name, such as masking-sporopollenin or abbreviated to masking-spn.     

Conserved metabolic steps for sporopollenin precursor formation in tobacco and rice

The development of pollen wall with proper sporopollenin deposition is essential for pollen viability and male fertility in flowering plants. Sporopollenin is a complex biopolymer synthesized from fatty acid and phenolic derivatives. Recent investigations in Arabidopsis have identified a number of anther‐specific genes involved in the production of fatty‐acyl monomers potentially required for exine formation. The existence of ancient biochemical pathways for sporopollenin biosynthesis has been widely proposed but experimental evidence from plant species other than Arabidopsis is not extensively available. Here, we investigated the metabolic steps catalyzed by the anther‐specific acyl‐CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide α‐pyrone reductase (TKPR). Using fatty acids as starting substrates, sequential activities of heterologously expressed tobacco enzymes NtACOS1, NtPKS1 and NtTKPR1 resulted in the production of reduced tetraketide α‐pyrones. Transgenic RNA interference lines were then generated for the different tobacco genes which were demonstrated to be indispensable for normal pollen development and male fertility. Similarly, recombinant rice OsPKS1 and OsTKPR1 were shown to function as downstream enzymes of NtACOS1. In addition, insertion mutant lines for these rice genes displayed different levels of impaired pollen and seed formation. Taken together, reduced tetraketide α‐pyrones appear to represent common sporopollenin fatty‐acyl precursors essential for male fertility in taxonomically distinct plant species.     

Indicative value ofPediastrum and other coccal green algae in palaeoecology

Sporopollenin layers in the cell wall of coccal green algae are responsible for the resistance of cell walls to destructive processes during fossilization as well as during chemical preparation of samples for pollen-analysis. Pollen slides of samples from limnic sediments thus also contain some algal cell walls. Although some pollen-analysts tried to stress this fact, the finds of algae in pollen slides have not been paid systematic attention yet, despite their potential use for a more accurate palaeoecological reconstruction. The article summarizes the results of palaeoecological studies showing how the algae can be used in palaeoecological reconstruction of past environments. The possibility of utilizing the indicative value of algal finds is demonstrated on examples of algal communities from fossil, subrecent and recent sediments from different longitudes, latitudes, and altitudes. The identification and indicative values of species and varieties ofPediastrum are included in a special review (Komárek &; Jankovská, Biblioth. Phycol., in press). The contemporary knowledge of ecological requirements of the given taxa, completed by information from their fossil finds, makes possible the reconstruction of trophic and temperature conditions and of the purity of the water environment of the past water biotopes.