Antioxidant properties of the pollen exine polymer matrix

The antioxidant properties of the polymer matrix of exine, the outer layer of pollen grain wall, were studied. The main component of this matrix is sporopollenin, a unique biopolymer resistant to mechanical and chemical damage. Samples of isolated exine purified from soluble compounds were studied with EPR using a stable nitroxyl radical TEMPO and a spin trap DMPO. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduced TEMPO and eliminated the hydroxyl radical. Also, fluorimetric analysis demonstrated that exine decomposed H₂O₂, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1 M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4 M NaOH, 170°C) of ether bonds, a large amount of hydroxycinnamic acids was released, and the exine almost completely lost its antioxidant capacity. The obtained results point to the ability of the extracellular polymer matrix of the exine to eliminate free radicals and H₂O₂ during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.     

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.     

Characterization and biosynthesis of non-degradable polymers in plant cuticles

The structure and monomeric composition of the highly aliphatic and non-saponifiable fraction of cutans isolated from the leaf cuticles of Agave americana L. and Clivia miniata Reg. have been elucidated. Spectroscopic Fourier transform infrared and ¹³C-nuclear magnetic resonance, calorimetric and X-ray diffraction studies, together with biopolymer analysis after exhaustive ozonolysis, showed that the cutan fraction consists of an amorphous three-dimensional network linked by ether bonds containing double bonds and free carboxylic acid functions. Data obtained from fatty acid sorption indicated that the new biopolymer investigated here has a highly hydrophobic character constituting an additional barrier biopolymer in those cuticles where it is present. Labelled [¹⁴C]linoleic acid was preferentially incorporated into the non-ester part of C. miniata leaf disks in comparison with the cutin fraction of the cuticular membrane. This indicates that the cis-pentadiene system of polyunsaturated fatty acids is involved in the formation of intramolecular linkages, mainly ether bonds, of the aliphatic biopolymer. Received: 29 June 1998 / Accepted: 17 September 1998     

Antioxidant properties of the pollen exine polymer matrix

The antioxidant properties of exine polymer matrix which forms the outer layer of pollen grain wall were studied. The main component of this matrix is sporopollenin - a unique biopolymer resistant to mechanical and chemical damage. The samples of isolated exine, purified from soluble compounds, were studied with EPR using stable nitroxyl radical TEMPO and DMPO spin trap. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduces TEMPO radical and eliminates hydroxyl radical. Also, the fluorometric analysis demonstrated that the exine eliminated H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4M NaOH, 170 degrees C) of ethers bonds a large amount of hydroxycinnamic acids has been released, and exines have lost their antioxidant capacity almost completely. The obtained results point to the ability of extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.     

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.     

Sporopollenin monomer biosynthesis in arabidopsis

Land plants have evolved aliphatic biopolymers that protect their cell surfaces against dehydration, pathogens, and chemical and physical damage. In flowering plants, a critical event during pollen maturation is the formation of the pollen surface structure. The pollen wall consists essentially of the microspore-derived intine and the sporophyte-derived exine. The major component of the exine is termed sporopollenin, a complex biopolymer. The chemical composition of sporopollenin remains poorlycharacterized because it is extremely resistant to chemical and biological degradation procedures. Recent characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated that the sporopollenin polymer consists of phenolic and fatty acid-derived constituents that are covalently coupled by ether and ester linkages. This review illuminates the outlines of a biosynthetic pathway involved in generating monomer constituents of the sporopollenin biopolymer component of the pollen wall.     

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.     

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.     

THE ULTRASTRUCTURE OF PHYCOPELTIS (CHROOLEPIDACEAE: CHLOROPHYTA). I. SPOROPOLLENIN IN THE CELL WALLS

Electron microscope observations on Phycopeltis epiphyton, a subaerial green alga found growing on the leaves of vascular plants and bryophytes, revealed the presence of a densely staining material within the inner and outer zones of the cell walls. This material resists acetolysis, is degraded by chromic acid, is unaffected by ethanolamine and exhibits secondary fluorescence when stained with the fluorochrome Primuline. These characteristics, together with infrared absorption spectra indicate that, on the basis of currently accepted criteria, the densely staining material is a sporopollenin and that it is a major component of the cell wall. Tests for cellulose, chitin, and lignin were negative, and little if any silica is present. It is suggested that negative results in tests for cellulose may be due to a masking effect by the sporopollenin. Comparison of the fine structure of the cell walls of P. epiphyton, pollen grains, and algal cells (known to contain sporopollenin) supports the suggestion that sporopollenin deposition on “unit membranes” is universal. Morphological similarity among sporopollenin lamellae in P. epiphyton, pollen grains, spores of land plants, and the trilaminar sporopollenin sheath in Chlorella, Scenedesmus, and Pediastrum indicates that the structures may be analogous. As in pollen grains, sporopollenin may provide protection against desiccation and parasitism. It may also be involved in the adhesion of Phycopeltis to host plants and in the adhesion between adjacent filaments of the thallus.     

(Poly)phenolic compounds in pollen and spores of Antarctic plants as indicators of solar UV-B – A new proxy for the reconstruction of past solar UV-B?

The morphology, size and characteristics of the pollen of the plant species Antarctic hairgrass (Deschampsia antarctica, Poaceae) and Antarctic pearlwort (Colobanthus quitensis, Caryophyllaceae) are described by scanning electron microscopy and light microscopy. Based on the number of pores the pollen of Colobanthus quitensis is classified as periporate or polypantorate, while that of Deschampsia antarctica is monoporate.

Pollen of Vicia faba plants, exposed to enhanced UV-B (10.6 kJ m^?2 day^?1 UV-B_BE) in a greenhouse, showed an increased content of UV-B absorbing compounds. There was also an increase of UV-B absorbing compounds in response to exposure to UV-A. By sequential chemical extraction three `compartments' of UV-B absorbance of pollen can be distinguished: a cytoplasmic fraction consisting of, e.g., flavonoids (acid-methanol extractable), a wall-bound fraction, consisting of, e.g., ferulic acid (NaOH extractable) and aromatic groups in the bioresistant polymer sporopollenin possibly consisting of, e.g., para-coumaric acid monomers (fraction remaining after acetolysis). The sporopollenin fraction in the pollen of Helleborus foetidus showed considerable UV-B absorbance (280–320 nm). There is evidence that enhanced solar UV-B induces increased UV-B absorbance (of sporopollenin) in pollen and spores of mosses, which may be preserved in the fossil record. As there are no instrumental records of solar UV-B in the Antarctic before 1970 and no instrumental records of stratospheric ozone over the Antarctic before 1957, the use of UV-B absorbing polyphenolics in pollen (and spores) as bio-indicator, or proxy of solar UV-B, may allow reconstruction of pre-ozone hole and subrecent UV-B and stratospheric ozone levels. Pollen and spores from herbarium specimens and from frozen moss banks (about 5000–10?000 years old) in the Antarctic may, therefore, represent a valuable archive of historical UV-B levels.

     

Botanical and geographical origin of honey from the dry and humid Chaco ecoregions (Argentina)

To characterise the botanical and geographical origin of honey from the Chaco region (Argentina), 189 samples of honey were examined for pollen content using standard methodology. A general feature was the high representation of pollen from native arboreal plants, mainly of Fabaceae and the scarcity of pollen from herbaceous plants. One hundred and twenty-three samples proved to be unifloral. The most common were from: Prosopis alba, Helianthus annuus, Sarcomphalus mistol, Eugenia uniflora and Schinopsis balansae followed by those of Copernicia alba, Trithrinax schizophylla, Tessaria integrifolia, Baccharis-Eupatorium, Cynophalla retusa, Schinopsis lorentzii, Gleditsia amorphoides, Heimia salicifolia, Pisonia zapallo, Sagittaria montevidensis and Bulnesia sarmientoi. The pollen spectrum allowed the Dry and Humid Chaco ecoregions to be distinguished. The presence of pollen from cultivated crops and naturalised plants reflects a transitional complex between both ecoregions.     

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Summary Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.     

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.     

Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map‐based cloning and sequence analysis, we identified a 1,459‐bp deletion in an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very‐long‐chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.     

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 POLLEN WALL OF CANNA AND ITS SIMILARITY TO THE GERMINAL APERTURES OF OTHER POLLEN

The pollen wall of Canna generalis Bailey is exceptionally thick, but only a minor part of it contains detectable amounts of sporopollenin. The sporopollenin is in isolated spinules at the exine surface and in the intine near the plasma membrane. There is no sporopollenin in the > 10 μ thick channeled region between spinules and intine. We suggest that the entire pollen wall of C. generalis is similar to the thick intine and thin exine typical for germinal apertures in many pollen grain types. Considered functionally, the Canna pollen wall may offer an infinite number of sites for pollen tube initiation and would differ significantly from grains that are inaperturate in the sense of an exine lacking definite germinal apertures.     

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.     

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.