Spore Exines Research Articles

A subclass II bHLH transcription factor in Marchantia polymorpha gives insight into the ancestral land plant trait of spore formation

Sporopollenin is often said to be one of the toughest biopolymers known to man. The shift in dormancy cell wall deposition from around the diploid zygotes of charophycean algae to sporopollenin around the haploid spores of land plants essentially imparted onto land plants the gift of passive motility, a key acquisition that contributed to their vast and successful colonization across terrestrial habitats.1,2 A putative transcription factor controlling the land plant mode of sporopollenin deposition is the subclass II bHLHs, which are conserved and novel to land plants, with mutants of genes in angiosperms and mosses divulging roles relating to tapetum degeneration and spore development.3,4,5,6,7 We demonstrate that a subclass II bHLH gene, MpbHLH37, regulates sporopollenin biosynthesis and deposition in the model liverwort Marchantia polymorpha. Mpbhlh37 sporophytes show a striking loss of secondary wall deposits of the capsule wall, the elaters, and the spore exine, all while maintaining spore viability, identifying MpbHLH37 as a master regulator of secondary wall deposits of the sporophyte. Localization of MpbHLH37 to the capsule wall and elaters of the sporophyte directly designates these tissue types as a bona fide tapetum in liverworts, giving support to the notion that the presence of a tapetum is an ancestral land plant trait. Finally, as early land plant spore walls exhibit evidence of tapetal deposition,8,9,10,11,12 a tapetal capsule wall could have provided these plants with a developmental mechanism for sporopollenin deposition.

Spore exines increase vitamin D clinical bioavailability by mucoadhesion and bile triggered release.

Sporopollenin exine capsules (SpECs) are microcapsules derived from the outer shells (exines) of plant spore and pollen grains. This work reports the first clinical study on healthy volunteers to show enhanced bioavailability of vitamin D encapsulated in SpECs from Lycopodium clavatum L. spore grains vs vitamin D alone, and the first evidence (in vitro, ex vivo and in vivo) of mechanisms to account for the enhancement and release of the active in the small intestine. Evidence for mucoadhesion of the SpECs contributing to the mechanism of the enhancement is based on: (i) release profile over time of vitamin D in a double blind cross-over human study showing significant release in the small intestine; (ii) in vivo particle counting data in rat showing preferred retention of SpECs vs synthetic beads; (iii) ex vivo99mTc labelling and counting data using rat small intestine sections showing preferred retention of SpECs vs synthetic beads; (iv) in vitro mucoadhesion data. Triggered release by bile in the small intestine was shown in vitro using solid state NMR and HPLC.

Unraveling the distinct germination processes of sporopollenin-based pollen grains and spores through morphological analyses upon natural nano-architectonics process

The outermost exine capsules of many pollen and spore grains are composed of a chemically inert yet mechanically robust sporopollenin biopolymer. These dynamically expansible and foldable capsules have great potential as renewable functional biomaterials with industrial applications. However, the mechanical and morphological variations in the shape, size, robustness, and apertural strength of the exine capsules across taxa of angiosperms and cryptogamic plants remains poorly understood. Thus, in this study, we unraveled the abortive microgel transformation of spores inspired by their germination mechanism, being compared with eudicot-based pollen microgels. After chemical treatments, significant mechanical degradation of exine was clearly observed for the Camellia pollen, whereas crosslinking density and modulus of spore exine remained almost constant. The significant volume expansion of Camellia pollen was observed akin to sunflower pollen; in contrast, the spores ballooned showed limited volume changes under equal levels of turgor pressure. Furthermore, spore underwent marked changes in volume when their aperture sutures were softened and ruptured, which are prerequisites for spore germination. Therefore, this study disentangled mechanical and morphological origins of biochemical pathways of pollen and spore germination, and germination-like hydration and desiccation, which will give clues about selection of pollen and spore species for potential biomaterial applications.

Fissures and pores in the capsule walls and hydrophobic elaters in Haplomitrium: a transmission and cryo-scanning electron microscope study

ABSTRACTIntroduction. Whereas the capsule wall in liverworts is usually described as smooth, our discovery of deep schizogenous fissures in the unistratose walls in Haplomitrium appears unique to this earliest diverging liverwort genus and may have important implications for understanding the evolution of the stomatal toolkit in land plants.Methods. Sporophytes of Haplomitrium gibbsiae and H. mnioides were examined by light, transmission and cryo-scanning electron microscopy.Key results. In striking contrast to cell breakage along the dehiscence grooves in the capsule walls, development of the schizogenous fissures is predetermined by a middle lamellar region of the radial walls comprising globular and fibrillar elements. The capsule wall cells remain alive, even after dehiscence, and have many cytological features in common with food-conducting cells in bryophytes including highly differentiated plasmodesmata. Whereas the highly ornamented spore exine retains water, the elaters are hydrophobic due to a coating of lipid droplets secreted by these cells.Conclusions. The development of regular thickened bands together with the schizogenous splitting of the capsule walls suggests that Haplomitrium may contain a possible primeval structural toolkit facilitating capsule desiccation and spore discharge. Thus we may be seeing, in this earliest extant liverwort lineage, the basic features needed for the assembly of stomata. There is no cytological evidence for a possible nutritive tapetal function for the capsule walls in Haplomitrium and the hydrophobic envelopes of the elaters probably enhance their effectiveness in spore dispersal.

Mimicking pollen and spore walls: self-assembly in action.

Decades of research have attempted to elucidate the underlying developmental mechanisms that give rise to the enormous diversity of pollen and spore exines. The organization of the exine starts with the establishment of an elaborate glycocalyx within which the subsequent accumulation of sporopollenin occurs. Ontogenetic studies using transmission electron microscopy of over 30 species from many different groups have shown that the sequence of structures observed during development of the exine corresponds to the sequence of self-assembling micellar mesophases (including liquid crystals) observed at increasing concentrations of surfactants. This suggested that self-assembly plays an important part in exine pattern determination. Some patterns resembling separate layers of spore and pollen grain walls have been obtained experimentally, in vitro, by self-assembly. However, to firmly establish this idea, columellate and granulate exines, the most widespread forms, needed to be simulated experimentally. We used our original method, preparing mixtures of substances analogous to those known to occur in the periplasmic space of developing microspores, then leaving the mixtures undisturbed for specific periods of time to allow the process of self-assembly to occur. We developed our method further by using new substances analogous to those present in the periplasmic space and performing the experiments in a thin layer, more closely resembling the dimensions of the periplasmic space. The artificial microstructures obtained from our in vitro self-assembly experiments closely resembled the main types of exines, including tectate-columellate, granulate, alveolate and structureless, and permitted comparison with both developing and mature microspore walls. Compared with the previous attempts, we managed to simulate columellate and granulate exines, including lamellate endexine. Our results show that simple physico-chemical interactions are able to generate patterns resembling those found in exines, supporting the idea that exine development in nature involves an interplay between the genome and self-assembly.

Leveraging plant exine capsules as pH-responsive delivery vehicles for hydrophobic nutraceutical encapsulation.

Plant exine capsules are natural microscale capsules that are highly physically robust and chemically resilient. They are extracted from pollen grains and plant spores, and can be used as renewable and safe microcapsules for encapsulation applications. Herein, we report the successful evacuation of natural sunflower pollen grains (SPGs) and Lycopodiastrum casuarinoides spore exine capsules (SECs) and investigated the effects of different loading methods on the encapsulation and release of nobiletin, a model hydrophobic nutraceutical. The compositional and morphological characterizations of SPGs and SECs obtained by the developed extraction protocol confirmed that the inside contents had been removed and hollow pollen cavities were obtained successfully. In addition, coumarin-6, a hydrophobic fluorescent probe, was encapsulated into SPGs and SECs, which proved the possibility of using them to encapsulate hydrophobic nutraceuticals. Furthermore, controlled release in simulated digestive fluids (SDF) was achieved by coating calcium alginate onto the outside of the SPGs and SECs. Looking forward, this may provide an effective and potential delivery model to protect loads from degradation in the stomach and achieve pH-responsive delivery of hydrophobic nutraceuticals after oral administration.

β-Carotene autoxidation: oxygen copolymerization, non-vitamin A products, and immunological activity

Carotenoids are reported to have immunological effects independent of vitamin A activity. Although antioxidant activity has been suggested as a basis of action, the ability of carotenoids to autoxidize to numerous non-vitamin A products with immunological activity is an alternative yet to be fully explored. We have undertaken a systematic study of β-carotene autoxidation and tested the product mixture for immunological activity. Autoxidation proceeds predominantly by oxygen copolymerization, leading to a defined, reproducible product corresponding to net uptake of almost 8 molar equivalents of oxygen. The product, termed OxC-beta, empirical formula C40H60O15versus C40H56for β-carotene, contains more than 30% oxygen (w/w) and 85% β-carotene oxygen copolymers (w/w) as well as minor amounts of many C8−C18 norisoprenoid compounds. No vitamin A or higher molecular weight norisoprenoids are present. The predominance of polymeric products has not been reported previously. The polymer appears to be a less polymerized form of sporopollenin, a biopolymer found in exines of spores and pollen. Autoxidations of lycopene and canthaxanthin show a similar predominance of polymeric products. OxC-beta exhibits immunological activity in a PCR gene expression array, indicating that carotenoid oxidation produces non-vitamin A products with immunomodulatory potential.

Viability of plant spore exine capsules for microencapsulation

Download options Please wait... Article information DOI https://doi.org/10.1039/C0JM02246B Article type Paper Submitted 12 Jul 2010 Accepted 07 Oct 2010 First published 12 Nov 2010 Download Citation J. Mater. Chem., 2011,21, 975-981 BibTex EndNote MEDLINE ProCite ReferenceManager RefWorks RIS Permissions Request permissions Viability of plant spore exine capsules for microencapsulation S. Barrier, A. Diego-Taboada, M. J. Thomasson, L. Madden, J. C. Pointon, J. D. Wadhawan, S. T. Beckett, S. L. Atkin and G. Mackenzie, J. Mater. Chem., 2011, 21, 975 DOI: 10.1039/C0JM02246B To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page. Read more about how to correctly acknowledge RSC content. Search articles by author Sylvain Barrier Alberto Diego-Taboada Matthew J. Thomasson Leigh Madden Joanna C. Pointon Jay D. Wadhawan Stephen T. Beckett Stephen L. Atkin Grahame Mackenzie

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family.

Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene. Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4.1b10). Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides. This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.

Simulated Self-assembly of Spore Exines

The spores and pollen of higher plants have enormous value in taxonomic studies by contributing to our understanding of past and present biodiversity, and of environmental change. The critical features used in species identification are wall structure and surface pattern. However, ultrastructural and histo-developmental studies of spore and pollen walls have, so far, provided limited explanation of wall construction and surface pattern formation. The consistency of pattern form within any species suggests a high degree of genetic regulation, and yet few templates or other mechanisms of control have been demonstrated. Our experiments show that all layers and organizations within the spore wall of our test plant group (lycopodiopsid megaspores) can be simulated by the flocculation of mixed colloidal systems. This leads us to a possible explanation of the mode of genetic control over pattern formation. It also provides a feasible, largely self-assembling, mechanism of construction which has the potential to reflect the diversity of structure known to exist in all spore and pollen walls.

The application of soft X-ray microscopy to the in-situ analysis of sporinite in coal

Soft X-ray imaging and carbon near edge absorption fine structure spectroscopy (C-NEXAFS) has been used for the in-situ analysis of sporinite in a rank-variable suite of organic-rich sediments extending from up to high volatile A bituminous coal. The acquisition of chemically based images (con trast based on the 1s−1π∗ transition of unsaturated carbon), reveals a homogeneous chemical structure in the spore exine. C-NEXAFS microanalysis indicates a chemical structural evolution of the sporopollenin bio/geopolymer with increasing maturation. The most significant change in the C-NEXAFS spectrum is an increase in unsaturated carbon, presumably aromatic, with rank. The rate of “aromatization” in sporinite in coal exceeds that of the associated vitrinite. Increases in the concentration of unsaturated carbon are paralleled by losses of aliphatic and hydroxylated aliphatic carbon components. Carboxyl groups are present in low and variable concentrations. Absorption due to carboxyl persists in the most mature specimen in this series, a high volatile A bituminous coal. The reactions that drive sporopollenin chemical structural evolution during diagenesis presumably involve sequential dehydration, Diels-Alder cyclo-addition, and dehydrogenation reactions that ultimately lead to a progressively aromatized bio/geopolymer.

13C solid-state n.m.r. spectroscopy of fossil sporopollenins. Variation in composition independent of diagenesis

13C solid-state nuclear magnetic resonance of sporopollenin from extant and extinct lycopods and gymnosperms shows that this material differs in composition between these major groups. The relative amounts of unsaturated carbon species are lower in the gymnosperms than in the lycopods in both the modern and fossil material. This suggests that the proportion of unsaturated carbon species present in the fossils is related to that of the original material. Since the fossil material used in this study was obtained from single rock samples, this largely eliminates the possibility that the observed differences in n.m.r. characteristics in fossil spore exines from different species can be attributed to different diagenetic histories.

Carbon-13 Solid-state Nuclear Magnetic Resonance of Sporopollenins from Modern and Fossil Plants

13 C solid-state nuclear magnetic resonance (NMR) has been applied to modern pollen and spore exines (Betula, Pinus and Lycopadium) and those of two fossil spores (Lagenicula and Parka) in order to assess the composition of their constituent sporopollenin. While they prove to have broadly similar structural characteristics, there are some significant differences between all types and particularly between the fossil and living material

A comparison of new microscopical techniques for the study of fossil spore wall ultrastructure

The two relatively new techniques of Scanning Acoustic Microscopy (SAM) and Laser Scanning Microscopy (LSM) are investigated for their potential use in studying fossil megaspore ultrastructure. Two Carboniferous ( Lagenicula and Setosisporites) and one Triassic megaspore ( Bowenispora) are used as examples. Comparisons of the conventional techniques of transmitted light, scanning and transmission electron microscopy are given. SAM and LSM provide useful additional tools for obtaining data on wall organisation. LSM particularly, provides good, medium magnification images of the spore exine.

The evolution of angiospermid pollen characteristics: conjectures and queries

The origin and evolution of different exine layers of palynofossils is analysed in the light of accumulating ultrastructural data. Semi-diagramatic illustrations based on the published TEM results of various exine types representing a cross section of extinct and extant plant groups are given for easy reference and comprehension. Some of the important palynological questions and issues discussed in the present work are imprecise use to describe the infra-tectum of pollen, of the flexible term "granular" that often leads to erroneous derivations and conclusions; ontogenetic differences between the apparently similar complexly alveolate columellate sexine types of gymnosperms and angiosperms respectively; independent evolution of columellar complexity in unrelated taxa: role of ubiquitous white lines in the exines of extinct and extant spores and pollen and adaptive rather than phylogenetic significance of sacci in progymnosperms, gymnosperms and angiosperms. Despite recognition of angiospermid pollen characteristics especially in tectally reticulate and columellate pollen of Triassic (Cornet, 1979, 1985, 1989; Pocock & Vasanthy, 1988; Pocock, Vasanthy & Venkatachala. 1988) the pre-Cretaceous origin of angiosperm still remains an open question.

Apical Organization and Leaf Ontogeny in Haplomitrium mnioides (Lindb.) Schust.

In nature, the vegetative shoots of Haplomitrium mnioides (Lindb.) Schust. are prostrate and display a novel pattern of anisophylly in which the reduced row of leaves is dorsal rather than ventral, while the fertile shoots are erect and nearly isophyllous. In both cases, the leaves are typically entire and subrotund to broadly ovate. When grown on moistened vermiculite in environmental chambers, however, the vegetative shoots assume an erect, nearly isophyllous habit, and, along with the fertile shoots, produce bifid or incised leaves. Both normal prostrate and cultureinduced erect shoots were studied using serial sections and scanning electron microscopy (SEM) in order to determine if changes in apical organization are correlated with these changes in habit. A tetrahedral apical cell with nearly equal cutting faces was found to be responsible for growth in both types of shoots and the apical derivatives were found to divide as in the Metzgeriales so that but a single leaf initial is formed per merophyte. Thus, in both the normal entire and culture-induced divided leaves, initial growth is by a single depressed-ovoid apical cell, which in the bifid leaves eventually cuts itself in two. The lateral projections of the incised leaves each develop from a cell on the margin of an already expanded leaf. In anisophyllous shoots, cell divisions in the developing dorsal leaves are arrested at an earlier stage than in the lateral ones. In basic patterns of apical organization and leaf ontogeny, H. mnioides resembles H. hookeri (Smith) Nees and H. blumei (Nees) Schust., and all three appear in these characters to be more closely aligned with the Metzgeriales than with the Jungermanniales or Takakiophyta. Haplomitrium Nees has long been the subject of taxonomic and phylogenetic speculation because of its peculiar array of characteristics, both those unique to the genus and those which are superficially indicative of other taxa, particularly the Jungermanniales, the Metzgeriales, and Takakia Hatt. & Inoue. Although Haplomitrium superficially resembles the leafy hepatics, it has recently been postulated to be more closely related to the Metzgeriales because it is anacrogynous, forms similar preprophase spore exine precursors (Brown & Lemmon 1986), and has been reported to possess a pattern of apical derivative development and leaf ontogeny like members of the Metzgeriales (Leitgeb 1875; Goebel 1891; Campbell 1920; Schuster 1966a, b; Crandall-Stotler 1981). Apical organization and leaf ontogeny have been discussed by Gottsche (1843) and Leitgeb (1875) for H. hookeri (Smith) Nees, an isophyllous species, while Goebel (189 1) and D. H. Campbell (1920) did the same for the anisophyllous Calobryum blumei Nees (=H. blumei (Nees) Schust. [Schuster 1963]). Although E. O. Campbell (1959) did not outline the stages of leaf development, she did discuss the apical organization and alluded to leaf origin in the isophyllous Calobryum gibbsiae Steph. (=H. gibbsiae (Steph.) Schust. [Schuster 1963]). In each case, plants p ssessing only the entire leaves typical of Haplomitrium were studied. Culture-induced bifid and ncised leaves have been reported in H. mnioides (Lindb.) Schust. by Kitagawa (1984); however, their ontogeny has not been described. The discovery of these divided leaves, which are superficially indicative of the Jungermanniales, raises the question as to whether more than one developmental pattern exists for the leaves of Halomitrium. As pointed out by Renzaglia (1982), the merophyte division pattern which is responsible for leaf formation is fundamentally different in metzgerialian and jungermannialian taxa. If the dissected leaves of H. mnioides depart from the metzgerialian onitogenetic pattern, in which but a single leaf initial is formed, and instead, develop from two initials as do jungermannialian leaves, that Haplomitrium is intermediate between the Jungermanniales and the Metzgeriales would be suggested. Clarification of these ontogenetic patterns is obviously essential to determine the position that Haplomitrium occupies in liverwort phylogeny. To this end, this study was undertaken. MATERIALS AND METHODS Live H. mnioides plants were obtained from Dr. M. Mizutani, collected 10 December 1985, from Kyushu, Mi0007-2745/87/383-389$0.85/0 This content downloaded from 157.55.39.59 on Mon, 17 Oct 2016 04:42:53 UTC All use subject to http://about.jstor.org/terms 384 THE BRYOLOGIST [VOL. 90

Sporocytic Control of Spore Wall Pattern in Liverworts

Extracellular material preferentially deposited in the sporocyte wall during meiotic prophase serves as an exine precursor in development of the distinctively patterned spore exines of the liverworts Haplomitrium hookerii and Pallavicinia lyellii. This unusual occurrence of prepat-terning of the exine in prophasic sporocytes provides clear structural evidence that control of wall patterning is an integral part of a genetic program triggered in the sporocyte and, at least in some cases, does not depend upon genetic transcription in the haploid nucleus following meiosis.

SPOROCYTIC CONTROL OF SPORE WALL PATTERN IN LIVERWORTS

Extracellular material preferentially deposited in the sporocyte wall during meiotic prophase serves as an exine precursor in development of the distinctively patterned spore exines of the liverworts Haplomitrium hookerii and Pallavicinia lyellii. This unusual occurrence of prepat‐terning of the exine in prophasic sporocytes provides clear structural evidence that control of wall patterning is an integral part of a genetic program triggered in the sporocyte and, at least in some cases, does not depend upon genetic transcription in the haploid nucleus following meiosis.

Spore wall development in Sphagnum lescurii

The spore wall of Sphagnum is unique in the Bryophyta. The Sphagnum spore exine consists of two layers: an inner, lamellate layer (A layer) and a thick, homogenous, outer layer (B layer). The exine of other mosses consists of only the outermost homogenous layer and, at most, a thin ill-defined opaque layer. During development of the A-layer exine and the intine, a cortical system of evenly spaced microtubules underlies the plasma membrane. The ontogeny of the wall layers is not strictly centripetal. The A-layer exine develops evenly around the young spore immediately after cytokinesis. As the intine is deposited centripetally inside it, the homogenous B-layer exine is deposited outside the first-formed A layer. The B layer is responsible for the primary sculpturing of the spore surface. The mature spore is covered by an outermost perine, which is responsible for secondary surface ornamentation. A trilaesurate aperture develops on the proximal surface of each spore after deposition of the A layer. Ridges of the laesurae develop as a result of deposition of thick areas of intine. The ridges are eventually covered by the outer wall layers, whereas the fissure is covered only by the A layer and a very thin B-layer exine. The complex development of the trilaesurate aperture is evidence that the structure is not merely a mechanically induced "trilete mark" or "scar" resulting from compression of tetrahedrally arranged spores within a sporocyte wall.

A model of exine substructure based on dissection of pollen and spore exines

Abstract The exine according to our model, consists of filamentous subunits (∼ 15–40nm in diam.), each containing an axially orientated tubule (∼ 10–15nm in diam.) with numerous lateral branches. Relatively few lateral branches are exposed at the surface of exinous subunits so that most of the polysaccharide‐, protein‐, and lipid‐containing tubular complex embedded within the sporopolleninous subunit is protected from degradation and unavailable to stains or ions that could cause molecules of the complex to swell or contract. In the intact exine, the few branches of the tubular complex (glycocalyx units) that are exposed at surfaces of subunits may affect exine size and staining, but their effect is not discernible from exogenous and nonstructural endogenous substances in the microcapillary space between exinous subunits. The existence of glycocalyx units is evident following treatment that etches the sporopollenin from exinous subunits (and eliminates exogenous and endogenous substances), without inactiv...