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.     

Chemical Constituents of Essential Oil of Two Varieties of Artemisia dubia Wall. ex Bess.

The essential oil of Artemisia dubia var. dubia and dubia var. subdigitata were analysed by GCMS and a tatal of 82 compounds including 50 terpenoids, 12 aromatics and 20 aliphatics were identified, among which variety dubia and var. subdigitata contained 54 and 67 compounds respectivey. Comparative studies indicate that 39 compounds (27 terpenoids, 5 aromatics and 7 aliphatics) were shared by the two varieties. The difference in constituents of essential oil between the two varieties is that more types of biocyclic and tricyclic sesquiterpenes, aromatics and aliphatics were shown inA. Dubia var. subdigitata than A. dubia var. dubia. Compositionally, the terpenoids in essential oil of the two varieties were endowed with a higher degree of cyclization than those of the species from Subgen. Artemisia, but similar to those of the species from Subgen. Dracunculus. Thus the assignment of the two varieties in Subgen. Dracunculus was consistent with the phylogeny of the genus Artemisia.     

牛尾蒿两变种精油的化学成分

本文以气相色谱质谱计算机联用的方法分析了牛尾蒿原变种Artemisia dubia var. dubia及无毛变种B A. dubia var. subdigitata精油化学成分,共鉴定出82种化合物,其中萜类50种,芳香类12种,脂肪族类20种。经比较研究表明,这两变种的精油中有39种化合物(27 种萜类,5种芳香类和7种脂肪族类化合物)相同,而它们的差别在于牛尾蒿无毛变种中二环、三环倍半萜、芳香类及脂肪族类化合物的类型比原变种多。从精油的萜类化学组成特点来看,这两个变种的萜类化合物环化程度比蒿亚属植物高,而与龙蒿亚属植物相近。因此,这两个变种归属于龙蒿亚属较合适。     

Acetylation and silylation of piperidine solubilized sporopollenin from pollen of Typha angustifolia L

Silyl and acetyl derivatives of sporopollenin from the pollen of Typha angustifolia L. were prepared. The derivatized products were readily soluble in piperidine-d11 and could be investigated employing one- and two-dimensional proton and carbon NMR (nuclear magnetic resonance) spectroscopy (1H,1H-COSY and 13C,1H-HETCOR techniques). For the first time, a two dimensional 13C,1H-HETCOR NMR spectrum of a sporopollenin could be obtained. The results underline the importance of derivatization techniques for obtaining two dimensional 13C-NMR spectra of sporopollenins. Moreover, piperidine turns out to be a more suitable solvent for sporopollenins than 2-aminoethanol, as it allows for higher solubilities, being important for 2D-NMR investigations. From the HETCOR and COSY spectra of the silylated and the acetylated Typha samples the occurrence of aliphatic polyhydroxy compounds as well as phenolic OH groups became evident.     

Evolutionary stasis of sporopollenin biochemistry revealed by unaltered Pennsylvanian spores

The biopolymer sporopollenin present in the spore/pollen walls of all land plants is regarded as one of the most recalcitrant biomacromolecules (biopolymers), providing protection against a range of abiotic stresses. This long-term stability is demonstrated by the near-ubiquitous presence of pollen and spores in the fossil record with spores providing the first evidence for the colonization of the land. Here, we report for the first time chemical analyses of geologically unaltered sporopollenin from Pennsylvanian (c. 310?million yr before present (MyBP)) cave deposits. Our data show that Pennsylvanian Lycophyta megaspore sporopollenin has a strong chemical resemblance to extant relatives and indicates that a co-polymer model of sporopollenin formation is the most likely configuration. Broader comparison indicates that extant sporopollenin structure is similar across widely spaced phylogenetic groups and suggests land plant sporopollenin structure has remained stable since embryophytes invaded land.     

Studies of Fossil and Modern Spore Wall Biomacromolecules using13C Solid State NMR

A range of Carboniferous lycophyte megaspore exines have beeninvestigated using¹³C magic-angle spinning nuclear magneticresonance (MAS NMR) spectroscopy. Their composition differsconsiderably from sporopollenin obtained from an extant lycophyte.The differences observed result in part from varying degreesof diagenesis. Fossil fern spores, gymnosperm megaspore-membranes and pollenhave also been examined. These show a similar composition tothe fossil lycophyte megaspores. The constituent material ofall of these exines differs considerably from the sporopolleninobtained from comparable extant samples. Despite the changesin composition observed on fossilisation, differences in compositionbetween the major groups of plants may be preserved to someextent in the fossil material. Walls of the fossil prasinophyceanalgal cystTasmanites have been examined and these show a greatersimilarity to fossil cuticle and algaenans than to sporopollenins. The effect of oxidative maceration on fossil and modern sporopolleninshas also been investigated. The main influence of oxidativemaceration is the removal of unsaturated carbon environmentssuch as aromatics; this causes fossil spores to be more susceptibleto oxidative maceration than the modern exines. Heating of modernexine material models the alteration of exines by diagenesis.The changes that occur on heating an extant sample to 150–225°Cgive a chemical composition that is similar to those of thefossil sporopollenins. ¹³C solid state NMR; spores; pollen; fossil; Carboniferous lycopsids; ferns; pteridosperm; gymnosperm; oxidative maceration; heating; thermal maturation     

FTIR characterisation of the chemical composition of Silurian miospores (cryptospores and trilete spores) from Gotland, Sweden

To better understand the biological affinities of cryptospores, micro-FTIR (Fourier transform infrared) spectroscopy analysis has been carried out on isolated specimens from the Upper Silurian of Gotland. The geobiochemical results have been compared to spectra of trilete spores, chitinozoans and leiospheres from the same sample. The palynomorphs are all very well preserved as attested by their pale yellow to orange colour indicative of a low thermal maturity. Micro-FTIR spectroscopy indicates that cryptospores display very similar spectra to those of the trilete spores, which are composed of sporopollenin characterised by absorption bands from aliphatic C-H in methylene (CH₂) and methyl (CH₃) groups, aromatic (C=C and C-H) groups and C=O groups of carboxylic acids. The sporopollenin composition of the cryptospore wall observed here is additional evidence demonstrating the embryophytic affinity of the cryptospores. In addition, several variations in other bands in the spectra of the different miospore morphospecies are evidenced and may be linked to their biological affinity or palaeoecological history.     

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.     

Pollen and flower volatiles in two Rosa species

GC/MS of headspace volatiles adsorbed separately from whole flowers and from pollen of R. rugosa shows distinct chemical profiles composed of aliphatics, terpenoids, and aromatics; only one-third of the identified compounds were detected in both samples. Analysis of pollenkitt extract indicates that most major pollen volatiles are included in this oily pollen coat. Volatiles from R. canina pollen contained few constituents in common with R. rugosa.     

Studies on sporopollenin biosynthesis in Tulipa anthers

Summary Extensive tracer experiments were carried out on Tulipa with the aim of determining the structure and biosynthesis of sporopollenin. The radiolabeled precursors were applied using an improved technique previously selected. The sporopollenin fraction was purified using either a gentle method — hydrolyzing enzymes (pronase, amylase, amyloglucosidase, cellulase, pectinase and lipase) and alkaline hydrolysis (method A) — or by a conventional aggressive procedure, where the material was enriched by alkaline hydrolysis and treated several days with 80% phosphoric acid (method B). The ¹⁴C-labeled precursors applied were mevalonate, glucose, acetate, malonic acid, phenylalanine, tyrosine, p-coumaric acid. Regardless of the method of enrichment, a higher level of incorporation into the sporopollenin fraction was always seen with [U-¹⁴C]-phenylalanine. The level of radioactivity found in sporopollenin labeled by phenylalanine or malonate was sufficiently high for the labeled polymer to be degraded and the products released analyzed for the first time. In the case of phenylalanine-labeled sporopollenin, the main degradation component, p-hydroxybenzoic acid, was also the most heavily labeled substance. This result was not dependent on the procedure used for sporopollenin enrichment. These findings are interpreted as meaning that phenylpropane metabolism via phenylalanine-ammonia lyase is involved in sporopollenin biosynthesis.     

Stable isotope ratios of carbon and nitrogen in pollen grains in order to characterize plant functional groups and photosynthetic pathway types

Measurements of delta(13)C, delta(15)N and C : N ratios on modern pollen grains from temperate plants, including whole grains as well as extracted sporopollenin, were analysed in order to characterize physiological plant types at the pollen level and to determine the variation of these parameters in modern pollen grains of the same climatic area. Measurements are presented for 95 batches of whole modern pollen from 58 temperate species and on the stable fraction of modern pollen grains, chemically extracted sporopollenin, for two modern species. Fourier transform infrared (FTIR) and cross-polarization and magic-angle spinning (CP/MAS) sporopollenin spectra were conducted in parallel. C(3) and C(4) plants can be separated by delta(13)C measurements based on pollen. Probabilistic assignments to plant functional groups (herbaceous, deciduous woody, evergreen woody) of C(3) plants by the means of a discriminant analysis can be made for C : N ratios and for delta(13)C. The results are related to other studies on sporopollenin in order to use this method in future work on fossil samples. Stable isotope measurements on pollen allow improved pollen diagrams, including forms that cannot be differentiated at species level, increasing the accuracy and resolution of plant physiological type distribution in quaternary and older fossil sediments.     

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.     

New insights from MALDI-ToF MS, NMR, and GC-MS: mass spectrometry techniques applied to palynology

Summary. The present study for the first time describes the application of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) to palynology. With an accessible mass range of up to about 350,000 Da at subpicomolar range, this technique is ideal for the characterisation of bio-macromolecules, such as sporopollenin, found in fossil and extant pollen and spore walls, which often can only be isolated in very small quantities. At this stage, the limited solubility of sporopollenin allows for the identification of sections of this biopolymer, but with the optimisation of MALDI-ToF matrices, further structure elucidation will become possible. Furthermore, gas chromatography–mass spectrometry (GC-MS) and ¹H nuclear magnetic resonance (¹H NMR) spectroscopy data obtained from a number of experiments revealed that some previously reported data were misinterpreted. These results add support to the hypothesis that common plasticizers were wrongly described as sporopollenin compounds. Correspondence and reprints: School of Earth, Ocean and Planetary Sciences, Cardiff University, Park Place, Cardiff CF10 3YE, United Kingdom. Permanent address: Albion College, Albion, Michigan, U.S.A.     

CYP704B1 Is a Long-Chain Fatty Acid ω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis

Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.The biopolymer sporopollenin is the major component of the outer walls in pollen and spores (exines). It is highly resistant to nonoxidative physical, chemical, and biological treatments and is insoluble in both aqueous and organic solvents. While the stability and resistance of sporopollenin account for the preservation of ancient pollen grains for millions of years with nearly full retention of morphology (Doyle and Hickey, 1976; Friis et al., 2001), these same qualities make it extremely difficult to study the chemical structure of sporopollenin. Thus, although the first studies on the composition of sporopollenin were reported in 1928 (Zetzsche and Huggler, 1928), the exact structure of sporopollenin remains unresolved. At present, it is thought that sporopollenin is a complex polymer primarily made of a mixture of fatty acids and phenolic compounds (Guilford et al., 1988; Wiermann et al., 2001).Fatty acids were first implicated as sporopollenin components when ozonolysis of Lycopodium clavatum and Pinus sylvestris exine yielded significant amounts of straight- and branched-chain monocarboxylic acids, characteristic fatty acid breakdown products (Shaw and Yeadon, 1966). More recently, improved purification and degradation techniques coupled with analytical methods, such as solid-state ¹³C-NMR spectroscopy, Fourier transform infrared spectroscopy, and ¹H-NMR, have shown that sporopollenin is made up of polyhydroxylated unbranched aliphatic units and also contains small amounts of oxygenated aromatic rings and phenylpropanoids (Guilford et al., 1988; Ahlers et al., 1999; Domínguez et al., 1999; Bubert et al., 2002). Biochemical studies using thiocarbamate herbicide inhibition of the chain-elongating steps in the synthesis of long-chain fatty acids and radioactive tracer experiments provided further evidence that lipid metabolism is involved in the biosynthesis of sporopollenin (Wilwesmeier and Wiermann, 1995; Meuter-Gerhards et al., 1999).Relatively little is known about the genetic network that determines sporopollenin synthesis. However, several Arabidopsis (Arabidopsis thaliana) genes implicated in exine biosynthesis encode proteins with sequence homology to enzymes that are involved in fatty acid metabolism. Mutations in MALE STERILITY2 (MS2) eliminate exine and affect a protein with sequence similarity to fatty acyl reductases; the predicted inability of ms2 plants to reduce pollen wall fatty acids to the corresponding alcohols suggests that this reaction is a key step in sporopollenin synthesis (Aarts et al., 1997). The FACELESS POLLEN1 (FLP1) gene, whose loss causes the flp1 exine defect, encodes a protein similar to those involved in wax synthesis (Ariizumi et al., 2003). The no exine formation1 (nef1) mutant accumulates reduced levels of lipids, and the NEF1 protein was suggested to be involved in either lipid transport or the maintenance of plastid membrane integrity, including those plastids in the secretory tapetum of anthers, where many of the sporopollenin components are synthesized (Ariizumi et al., 2004). The dex2 mutant has mutations in the evolutionarily conserved anther-specific cytochrome P450, CYP703A2 (Morant et al., 2007), which catalyzes in-chain hydroxylation of saturated medium-chain fatty acids, with lauric acid (C12:0) as a preferred substrate (Morant et al., 2007). A recently described gene, ACOS5, encodes a fatty acyl-CoA synthetase that has in vitro preference for medium-chain fatty acids (de Azevedo Souza et al., 2009). Mutations in all of these genes compromise exine formation.Here, we describe an evolutionarily conserved cytochrome P450, CYP704B1, and demonstrate that this gene is essential for exine biosynthesis and plays a role different from that of CYP703A2. Heterologously expressed CYP704B1 catalyzed ω-hydroxylation of several saturated and unsaturated C14-C18 fatty acids. These results suggest the possibility that ω-hydroxylated fatty acids produced by CYP704B1, together with in-chain hydroxylated lauric acids provided by the action of CYP703A2, may serve as key monomeric aliphatic building blocks in sporopollenin formation. Analyses of the genetic relationships between CYP704B1, MS2, and CYP703A2 suggest that all three genes are involved in the same pathway within the sporopollenin biosynthesis framework.     

Pollen sporopollenin: degradation and structural elucidation

We report the isolation of purified sporopollenin from pollen grains of different species and its complete solubilization. Exine from Pinus pinaster, Betula alba, Ambrosia elatior and Capsicum annuum was extracted by treatment with hydrogen fluoride in pyridine. These exines were purified from their aromatic moieties and from fatty acids linked by ester bonds using acidolysis and saponification treatments. The biopolymer obtained retains almost completely the shape of the original pollen grain. Fourier-transform infrared spectroscopy analysis of the isolated sporopollenin showed the absence of polysaccharide and phenolic material and the presence of carboxylic acid groups joined to unsaturations and ether linkages. Sporopollenin samples were successfully degraded by exhaustive 24-h ozonolysis at room temperature. Gentle ozonolysis (3 h at 0°C) did not completely degrade the biopolymer. The compounds obtained after exhaustive ozonolysis were analysed by gas chromatography-mass spectrometry. Dicarboxylic acids with a low number of carbon atoms were identified as major components of sporopollenin from P. pinaster, A. elatior and C. annuum, representing 28.8%, 63.2% and 88.5%, respectively, of the total compounds obtained. Fatty acids and n-alkanes also were identified in P. pinaster, A. elatior and B. alba sporopollenin. From the data obtained, an hypothesis about the chemical nature and structural arrangement of the sporopollenin is proposed. Received: 8 November 1998 / Revision accepted: 14 April 1999     

On Comparison Between Fungal and Bacterial Pretreatment of Coal for Enhanced Biogenic Methane Generation

Owing to better understanding of subsurface geochemical carbon recycling and real-time active methanogenesis in major coal basins around the globe, substantial share of subsurface methane generation is attributed to biogenic origin. Since coal, being complex geopolymer, does not appear to be a favorable microbial substrate, enhancement in biogenic methane yield depends on its degradation into simpler organic substrates. This review puts forward a comparative analysis of fungal and bacterial pretreatment for determining the extent of facilitation in initial degradation of coal, which is still rate limiting step in overall conversion of coal into methane. Primarily, the initial fungal degradation of coal differs from bacterial pretreatment of coal in terms of the nature of released organics. On the basis of previous reports, fungal pretreatment of coal yields, majorly, polyaromatic hydrocarbons, however, bacterial pretreatment results in the generation of mixed organics pool of aromatics and aliphatics. The presence of aliphatics may be prospected for achieving greater conversion rates of coal conversion into methane. Considering the criticality of preliminary degradation of coal and associated issues, the fate of commercial biogenic methane generation would be dictated by the factors pertaining to geological considerations and reservoir geology, chemistry of coal and associated water tables, geomicrobial considerations and economic viability.     

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.     

An analysis of the fur of river otters in Prince William Sound, Alaska: oil related hydrocarbons 8 years after the Exxon Valdez oil spill

Approximately 8 years after the Exxon Valdez oil spill, river otters (Lutra canadensis) were trapped from the shoreline in both oiled (Knight Island) and nonoiled (Jackpot Bay) areas of Prince William Sound, Alaska. Captive river otters were wiped with isopropanol-soaked gauze and the gauze extracts were analyzed by gas chromatography with mass spectrometry detection. Differences in pentacosane (C-25) levels in the fur were observed between the oiled and nonoiled sites, while lower molecular weight aliphatics and aromatics were absent. These data are useful when evaluating the role of fur grooming in the long-term exposure of river otters to hydrocarbons and the expression of P450-1A in Knight Island otters. Accepted: 26 August 1998     

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with 13C and determination and interpretation of terminal pK values

The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.