Protist-like inclusions in amber, as evidenced by Charentes amber

The mid-Cretaceous amber of France contains thousands of protist-like inclusions similar in shape to some ciliates, flagellates and amoebae. The sheer abundance of these inclusions and their size variation within a single amber piece are not concordant with true fossil protists. French amber is coniferous in origin, which generally does not preserve well protists without cell walls. Thus, it would be surprising if French Cretaceous amber had preserved millions of protists. Here, we present a survey of the protist-like inclusions from French amber and attempt to elucidate their origins.Diverse Cretaceous ambers (from Spain, Germany and Lebanon), also derived from conifer resins, contain thousands of protist-like inclusions. In contrast, Tertiary ambers and modern resins are poor in protist-like fossils. This suggests these inclusions originated from early Cretaceous plant resins, probably secreted with the resin by trees that did not survive after the Cretaceous (such as the Cheirolepidiaceae). A review of the recent literature on amber microfossils indicates several protist-like inclusions that are unlikely to have a biological origin have already been described as real fossil protists. This is problematic in that it will bias our understanding of protist evolution.     

The Range of Bioinclusions and Pseudoinclusions Preserved in a New Turonian (~90 Ma) Amber Occurrence from Southern Australia

A new Turonian amber occurrence, representing the oldest in situ amber locality in Australia and the southern-most locality in Gondwana, has recently been discovered in the Otway Basin of Victoria. The amber was collected from petroleum cores and many pieces contain a range of inclusions that can provide information on the depositional history of the resin. To date, one species of fern spore (Cyathidites minor) and one species of lycophyte spore (Kraeuselisporites sp?) have been conclusively identified in the amber, along with filamentous microorganisms and degraded plant matter. Several samples are also rife with pseudoinclusions as reported recently in other ambers. The abundance of preserved particulate debris and wind dispersed spores suggest that the Otway amber formed subaerially. Furthermore, based on the range of bioinclusions and forms of pseudoinclusions preserved within a single piece of amber, the locus of hardening for individual samples is variably interpreted as occurring in the tree tops, on the tree trunk or on the ground surface. Notably, specific inclusion assemblages are associated with certain colours of amber. By extension, and in accordance with recent studies, amber colour may be indicative of depositional environment. Variation in the environment of solidification may, therefore, be sufficient to account for the broad range of morphological characteristics preserved in a single amber deposit.     

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.     

Selective staining of fungal hyphae in parasitic and symbiotic plant-fungus associations

A cytochemical method for light microscopical studies is described which allows the specific detection of fungal hyphae in plant-fungus associations: e.g. lichens, mycorrhiza, or fungal infections of plant tissue. The specimens were fixed and embedded in epoxy resin by a standard protocol for electron microscopy. Semithin sections were successively incubated with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and calcofluor white (CW). FITC-WGA stained exclusively the fungal cell walls while CW stained both the fungal and the plant cell walls. Therefore, FITC-WGA is an excellent marker for the fungal hyphae.     

A microstructure study on silicified wood from the Permian Petrified Forest of Chemnitz

Three typical plant taxa from the fossil assemblage of the 290-million-year-old Chemnitz Petrified Forest (Zeisigwald Tuff Horizon, Leukersdorf Formation) were studied with regard to the microstructure of the petrifactions: samples of the tree fern Psaronius sp., the seed fern Medullosa stellata, and the gymnosperm Dadoxylon sp. The plant’s tissues are anatomically preserved by silica exhibiting different crystalline order and by other mineralisations. Specimens were studied by means of electron backscatter imaging and electron backscatter diffraction in a scanning electron microscope. The cell walls were largely preserved by quartz crystals, the cell lumina by cryptocrystalline silica. The former organisation and chemical composition of the vascular tissue are mirrored by varying grain formation and grain size. Results are discussed in terms of extant xylem cell wall organisation showing highly hydrophilic cellulose and hemicellulose cross-linked by hydrophobic lignin. The effect of polar and non-polar wood components on the precipitation of silica from aqueous solution and on the formation of crystals is convincing, and the reported results provide a better understanding of how silica replaced organic matter during the petrifaction process.     

Microbe‐like inclusions in tree resins and implications for the fossil record of protists in amber

During the past two decades, a plethora of fossil micro‐organisms have been described from various Triassic to Miocene ambers. However, in addition to entrapped microbes, ambers commonly contain microscopic inclusions that sometimes resemble amoebae, ciliates, microfungi, and unicellular algae in size and shape, but do not provide further diagnostic features thereof. For a better assessment of the actual fossil record of unicellular eukaryotes in amber, we studied equivalent inclusions in modern resin of the Araucariaceae; this conifer family comprises important amber‐producers in Earth history. Using time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), we investigated the chemical nature of the inclusion matter and the resin matrix. Whereas the matrix, as expected, showed a more hydrocarbon/aromatic‐dominated composition, the inclusions contain abundant salt ions and polar organics. However, the absence of signals characteristic for cellular biomass, namely distinctive proteinaceous amino acids and lipid moieties, indicates that the inclusions do not contain microbial cellular matter but salts and hydrophilic organic substances that probably derived from the plant itself. Rather than representing protists or their remains, these microbe‐like inclusions, for which we propose the term ‘pseudoinclusions’, consist of compounds that are immiscible with the terpenoid resin matrix and were probably secreted in small amounts together with the actual resin by the plant tissue. Consequently, reports of protists from amber that are only based on the similarity of the overall shape and size to extant taxa, but do not provide relevant features at light‐microscopical and ultrastructural level, cannot be accepted as unambiguous fossil evidence for these particular groups.     

Ectomycorrhizas from a Lower Eocene angiosperm forest

The development of mycorrhizal associations is considered a key innovation that enabled vascular plants to extensively colonize terrestrial habitats. Here, we present the first known fossil ectomycorrhizas from an angiosperm forest. Our fossils are preserved in a 52 million-yr-old piece of amber from the Tadkeshwar Lignite Mine of Gujarat State, western India. The amber was produced by representatives of Dipterocarpaceae in an early tropical broadleaf forest. The ectomycorrhizas were investigated using light microscopy and field emission scanning electron microscopy. Dissolving the amber surrounding one of the fossils allowed ultrastructural analyses and Raman spectroscopy. Approx. 20 unramified, cruciform and monopodial-pinnate ectomycorrhizas are fossilized adjacent to rootlets, and different developmental stages of the fossil mycorrhizas are delicately preserved in the ancient resin. Compounds of melanins were detectable in the dark hyphae. The mycobiont, Eomelanomyces cenococcoides gen. et spec. nov., is considered to be an ascomycete; the host is most likely a dipterocarp representative. An early ectomycorrhizal association may have conferred an evolutionary advantage on dipterocarps. Our find indicates that ectomycorrhizas occurred contemporaneously within both gymnosperms (Pinaceae) and angiosperms (Dipterocarpaceae) by the Lower Eocene.     

Silicification of bamboo (Phyllostachys heterocycla Mitf.) root and leaf

Bamboo is a silicon accumulating plant. In leaves, the major place of silicon (Si) deposition is the epidermis, with the highest concentration of Si in silica cells. In bamboo roots, the deposition of Si is found only in endodermal cell walls. The silicification of leaves and roots was examined in the economically important bamboo plant Phyllostachys heterocycla, using an environmental scanning electron microscope coupled with X-ray microanalysis, as well as gravimetric quantification. The content of Si on a dry weight basis measured by gravimetric quantification was 7.6% in leaves and 2.4% in roots, respectively. Moreover, quantification of EDX data showed high Si impregnation of the inner tangential endodermal walls. Si content in this part of the root endodermal cell walls was even higher than that in the outer leaf epidermal walls, where conspicuous deposition of Si often occurs in grass plants.     

Multi‐scale spatial heterogeneity of pectic rhamnogalacturonan I (RG–I) structural features in tobacco seed endosperm cell walls

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG–I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG–I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG–I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG–I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG–I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG–I glycan complexity in the context of cell‐wall architectures and in relation to cell‐wall functions in cell and tissue development.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

Putative ancient microorganisms from amber nuggets.

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified.     

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.     

Subcellular localization of silicon and germanium in grass root and leaf tissues by SIMS: evidence for differential and active transport

Silicon transport and incorporation into plant tissue is important to both plant physiological function and to the influence plants have on ecosystem silica cycling. However, the mechanisms controlling this transport have only begun to be explored. In this study, we used secondary ion mass spectrometry (SIMS) to image concentrations of Si in root and shoot tissues of annual blue grass (Poa annua L.) and orchard grass (Dactylis glomerata L.) with the goal of identifying control points in the plant silica uptake pathway. In addition, we used SIMS to describe the distributions of germanium (Ge); the element used to trace Si in biogeochemical studies. Within root tissue, Si and Ge were localized in the suberized thick-walled region of endodermal cells, i.e. the proximal side of endodermal cells which is in close association to the casparian strip. In leaves, Si was present in the cell walls, but Ge was barely detectable. The selective localization of Si and Ge in the proximal side of endodermal cell walls of roots suggests transport control is exerted upon Si and Ge by the plant. The absence of Si in most root cell walls and its presence in the cell walls of leaves (in areas outside of the transpiration terminus) suggests modifications in the chemical form of Si to a form that favors Si complexation in the cell walls of leaf tissue. The low abundance of Ge in leaf tissue is consistent with previous studies that suggest preferential transport of Si relative to Ge.     

Subcellular distribution of chromium in accumulating plant Leersia hexandra Swartz

The subcellular distribution of chromium in Leersia hexandra Swartz, a Cr-accumulating plant found in China, was studied by differential centrifugation, transmission electron microscope and energy dispersive analysis of X-ray. Subcellular fractionation of Cr-containing tissues showed that most of the accumulated Cr was isolated to the cell walls in roots and the vacuoles in leaves. When the plant was grown in a nutrient solution containing 60 mg L^?1 Cr, 83.2% of the root Cr was localized in the cell wall fraction, while 57.5% of leaf Cr was localized in the vacuole and cytoplasm fraction. Transmission electron microscopic analysis revealed that those cell compartments contained especially electron dense areas. Energy dispersive X-ray spectra showed the electron dense areas contained high Cr. However, the dark electron precipitates were never observed in the plant cells without Cr treatment. In all treatment groups (5, 30 and 60 mg L^?1), the fraction containing the lowest level of Cr was the organelle fraction in roots as well as leaves. These results indicated that Cr accumulated in the L. hexandra was preferentially stored in the cell walls of roots and the vacuoles of leaves. This phenomenon diverted Cr ions from metabolically active compartment (chloroplast, mitochondria), resulting in a reduction of Cr toxicity in the plant cell.     

Distribution of aluminium in accumulator plants by X-ray microanalysis in Richeria grandis Vahl leaves from a cloud forest in Venezuela

Abstract. The leaf cells of a tropical tree which accumulates more than 1mg per gram dry weight of Al in its leaves were studied by X-ray microanalysis. The analyses made in cell walls, vacuoles and chloroplasts of a mature leaf showed that A1 is accumulated preferentially in the cell wall. In the pre-senescent leaf, A1 also invades the interior of cells and is deposited in chloroplasts and vacuole. Aluminium was found also in xylem vessels but only in traces in phloem tissue. It was not found in spongy parenchyma cells. The observed A1 distribution, i.e. at the adaxial leaf face, coincides with observed damage in tissues of pre-senescent leaves. The means by which this plant copes with Al under natural conditions seems to be partially preventing it from entering the cell by deposition in cell walls. A hypothesis about the pathways of Al in accumulator plants is proposed.     

Penetration Behaviour of Venturia nashicola,Associated with Hydrogen Peroxide Generation,in Asian and European Pear Leaves

The penetration behaviour of the pathogen Venturia nashicola, which causes scab disease in Asian pears, was studied at the ultrastructural and cytochemical levels in host and non‐host leaves. We show, for the first time, that before V. nashicola penetrated the cuticle of the epidermis of the pear leaf, the appressorial bottom of the pathogen invaginated to form a cavity that contains electron‐dense material. The leaf cuticle beneath the cavity also became highly electron dense following penetration by V. nashicola. The location of these electron‐dense materials at the sites of penetration of the pathogen into plant cell walls suggests that they might be related to enzymes capable of degrading cell walls and that the cavities might be needed for successful penetration of leaves by V. nashicola. The generation of hydrogen peroxide (H₂O₂) was observed in penetration‐related infection structures of V. nashicola, such as appressorial bottoms, infection sacs, penetration pegs and necks of subcuticular hyphae regardless of whether the interaction of V. nashicola with pear plants was compatible or incompatible. Nonetheless, more H₂O₂ was generated at the sites of the structures in scab‐inoculated susceptible leaves than that in scab‐inoculated resistant ones. Furthermore, the decrease in the level of H₂O₂ generation following treatment with the antioxidant ascorbic acid partially prevented the penetration of the cuticle. Therefore, the generation of H₂O₂ from the penetration‐related structures might be a pathogenicity factor that contributes to strengthening the penetration peg of V. nashicola.     

A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata

Summary Plasmodesmata, dynamic pore structures that traverse plant cell walls, function in cytoplasmic transport between contiguous cells. Cell walls containing embedded plasmodesmata were isolated from mesocotyls of etiolated maize seedlings. Proteins associated with the isolated walls were separated by SDS-PAGE and antibodies were generated against a 41 kDa protein, one of several associated with this wall fraction. Immunoblot analysis showed that the 41 kDa polypeptide was also associated with other subcellular fractions obtained following tissue homogenization and differential centrifugation. The wall associated 41 kDa protein is apparently a peripheral membrane protein since it could be extracted by high salt and high pH. Silver-enhanced immunogold light microscopy showed that the 41 kDa protein was associated with the cell walls of cells both in the stele and cortex. The immunolabeling pattern was transwall and punctate. Electron microscopic immuno-gold labeling localized the polypeptide to plasmodesmata and to electron dense cytoplasmic structures that are apparently Golgi membranes. The significance of the presence of this protein in the Golgi is discussed.Abbreviations ER endoplasmic reticulum     

The fossil hornwort described from Dominican amber is an angiosperm flower

Hornworts (Anthocerotophyta) are a main lineage of land plants but they are exceedingly rare as fossils. The only fossil hornwort described from amber has been interpreted as the best preserved fossil of this group. Reinvestigation of this fossil revealed that this Miocene amber inclusion represents a poorly preserved flower that shows some features of the Caesalpinioideae subfamily of the Fabaceae.     

Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.