Molecular insights into the complex mechanics of plant epidermal cell walls.

Plants have evolved complex nanofibril-based cell walls to meet diverse biological and physical constraints. How strength and extensibility emerge from the nanoscale-to-mesoscale organization of growing cell walls has long been unresolved. We sought to clarify the mechanical roles of cellulose and matrix polysaccharides by developing a coarse-grained model based on polymer physics that recapitulates aspects of assembly and tensile mechanics of epidermal cell walls. Simple noncovalent binding interactions in the model generate bundled cellulose networks resembling that of primary cell walls and possessing stress-dependent elasticity, stiffening, and plasticity beyond a yield threshold. Plasticity originates from fibril-fibril sliding in aligned cellulose networks. This physical model provides quantitative insight into fundamental questions of plant mechanobiology and reveals design principles of biomaterials that combine stiffness with yielding and extensibility.

Tandem DNA repeats contain cis-regulatory sequences that activate biotrophy-specific expression of Magnaporthe effector gene PWL2.

During plant infection, fungi secrete effector proteins in coordination with distinct infection stages. Thus, the success of plant infection is determined by precise control of effector gene expression. We analysed the PWL2 effector gene of the rice blast fungus Magnaporthe oryzae to understand how effector genes are activated specifically during the early biotrophic stages of rice infection. Here, we used confocal live-cell imaging of M. oryzae transformants with various PWL2 promoter fragments fused to sensitive green fluorescent protein reporter genes to determine the expression patterns of PWL2 at the cellular level, together with quantitative reverse transcription PCR analyses at the tissue level. We found PWL2 expression was coupled with sequential biotrophic invasion of rice cells. PWL2 expression was induced in the appressorium upon penetration into a living rice cell but greatly declined in the highly branched hyphae when the first-invaded rice cell was dead. PWL2 expression then increased again as the hyphae penetrate into living adjacent cells. The expression of PWL2 required fungal penetration into living plant cells of either host rice or nonhost onion. Deletion and mutagenesis experiments further revealed that the tandem repeats in the PWL2 promoter contain 12-base pair sequences required for expression. We conclude that PWL2 expression is (a) activated by an unknown signal commonly present in living plant cells, (b) specific to biotrophic stages of fungal infection, and (c) requires 12-base pair cis-regulatory sequences in the promoter.

High-Resolution Imaging of Cellulose Organization in Cell Walls by Field Emission Scanning Electron Microscopy.

Field emission scanning electron microscopy (FESEM) is a powerful tool for analyzing surface structures of biological and nonbiological samples. However, when it is used to study fine structures of nanometer-sized microfibrils of epidermal cell walls, one often encounters tremendous challenges to acquire clear and undistorted images because of two major issues: (1) Preparation of samples suitable for high resolution imaging; due to the delicateness of some plant materials, such as onion epidermal cell walls, many things can happen during sample processing, which subsequently result in damaged samples or introduce artifacts. (2) Difficulties to acquire clear images of samples which are electron-beam sensitive and prone to charging artifacts at magnifications over 100,000×. In this chapter we described detailed procedures for sample preparation and conditions for high-resolution FESEM imaging of onion epidermal cell walls. The methods can be readily adapted for other wall materials.

Genotoxicity of the food additive E171, titanium dioxide, in the plants Lens culinaris L. and Allium cepa L.

E171 (titanium dioxide, TiO2), an authorized foods and beverage additive, is also used in food packaging and in pharmaceutical and cosmetic preparations. E171 is considered to be an inert and non-digestible material, not storable in animal tissues, but the possible presence of TiO2 nanoparticles (NP) may present a risk to human health and the environment. We determined the presence of 15% TiO2 NP in a commercial E171 food additive product, by electron microscopy. The biological effects of E171 were assessed in Lens culinaris and Allium cepa for the following endpoints: percentage of germination, root elongation, mitotic index, presence of chromosomal abnormalities, and micronuclei. The results indicated low phytotoxicity but dose-dependent genotoxicity. We also observed internalization of TiO2 NP and ultrastructural alterations in the root systems.

Iris yellow spot virus-induced chloroplast malformation results in male sterility.

Iris yellow spot virus (IYSV) is one of the most devastating viral pathogens, which causes high economic losses in the onion yield. Physiological and genetic changes are associated with the appearance of chlorotic symptom in the infected plants. IYSV-N gene sequence analysis revealed that it shared sequence identity of 99% with other Egyptian isolates, at both genomic and proteomic levels. In addition, N protein sequence with computational examination indicated many motifs involved and played different roles in the virus activity and its regulation and stability were detected. In the Differential Display-Polymerase Chain Reaction (DD-PCR) study, a highly up-regulated gene at 15 days post-biological IYSV inoculation (dpi) was selected for sequencing. Based on the sequencing results that showed the identified gene was coding for a chloroplast-related gene, degenerate specific primers were designed for Real-Time PCR analysis. A significant change in the transcription level of the chloroplast-related gene after 15 dpi suggested that some IYSV proteins interact and/or regulate with chloroplast proteins and this finding supports the DD-PCR results. At 20 dpi, the ultrathin sections showed that IYSV infection caused many dramatic chloroplasts malformations. The malformation appeared as chloroplast broken envelope with the presence of numerous spherical particles inside it and chloroplasts with long stromule. Our findings indicated that IYSV interrupts normal chloroplast functions, as a part of the onion defence response, however many crucial factors remain to be elucidated and further studies are needed at both biological and molecular levels.

Resonant soft X-ray scattering reveals cellulose microfibril spacing in plant primary cell walls.

Cellulose microfibrils are crucial for many of the remarkable mechanical properties of primary cell walls. Nevertheless, many structural features of cellulose microfibril organization in cell walls are not yet fully described. Microscopy techniques provide direct visualization of cell wall organization, and quantification of some aspects of wall microstructure is possible through image processing. Complementary to microscopy techniques, scattering yields structural information in reciprocal space over large sample areas. Using the onion epidermal wall as a model system, we introduce resonant soft X-ray scattering (RSoXS) to directly quantify the average interfibril spacing. Tuning the X-ray energy to the calcium L-edge enhances the contrast between cellulose and pectin due to the localization of calcium ions to homogalacturonan in the pectin matrix. As a consequence, RSoXS profiles reveal an average center-to-center distance between cellulose microfibrils or microfibril bundles of about 20 nm.

Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.

Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.

Fracture faces of frozen membranes: 50th anniversary.

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell's membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane's two external surfaces. During etching, when ice is allowed to sublime after fracturing, limited regions of the actual membrane surfaces are revealed. Examination of the fractured faces and etched surfaces provided strong evidence that biological membranes are organized as lipid bilayers with some proteins on the surface and other proteins extending through the bilayer. Membrane splitting made it possible for electron microscopy to show the relative proportion of a membrane's area that exists in either of these two organizational modes.

MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation.

Advances in nanotechnology have led to the large-scale production of nanoparticles, which, in turn, increases the chances of environmental exposure. While humans (consumers/workers) are primarily at risk of being exposed to the adverse effect of nanoparticles, the effect on plants and other components of the environment cannot be ignored. The present work investigates the cytotoxic, genotoxic, and epigenetic (DNA methylation) effect of MWCNT on the plant system- Allium cepa. MWCNT uptake in root cells significantly altered cellular morphology. Membrane integrity and mitochondrial function were also compromised. The nanotubes induced significant DNA damage, micronucleus formation and chromosome aberration. DNA laddering assay revealed the formation of internucleosomal fragments, which is indicative of apoptotic cell death. This finding was confirmed by an accumulation of cells in the sub-G0 phase of the cell cycle. An increase in CpG methylation was observed using the isoschizomers MspI/HpaII. HPLC analysis of DNA samples revealed a significant increase in the levels of 5-methyl-deoxy-cytidine (5mdC). These results confirm the cyto-genotoxic effect of MWCNT in the plant system and simultaneously highlight the importance of this epigenetic study in nanoparticle toxicity.

[Phytotoxicity of colloidal solutions of metal-containing nanoparticles].

Phytotoxicity of colloidal solutions of metal-containing nanoparticles (Ag, Cu, Fe, Zn, Mn) has been investigated using a standard Allium cepa (L.) test system. Toxicity of experimental solutions at the organism level was evaluated in terms of biomass growth of onion roots, and cytotoxicity was estimated by the mitotic index of root meristem cells. The colloidal solutions of metal nanoparticles inhibited the growth of Allium cepa (L.) roots due to their ability to penetrate into cells and interact with their components, and thus to inhibit mitosis. According to our results cytotoxicity of test solutions decreases in the following order: Cu > or = Zn > Ag > or = Fe. Solution of Mn-containing nanoparticles revealed physiological activity according to root growth reaction.

The effect of pre-incubation of Allium cepa L. roots in the ATH-rich extract on Pb uptake and localization.

The positive influence of anthocyanin (ATH) on toxic metal-treated plant material is well documented; however, it is still not explained if it is caused by changes in element absorption and distribution. Therefore, detailed analysis of the effect of the ATH-rich extract from red cabbage leaves on Pb uptake and localization at morphological, anatomical and ultrastructural level was the goal of this study. Two-day-old adventitious roots of Allium cepa L. (cv. Polanowska) were treated for 2 h with the aqueous solution of Pb(NO3)2 at the concentration of 100 µM with or without preliminary incubation in the anthocyanin-rich extract from Brassica oleracea L. var. capitata rubra leaves (250 µM, 3 h). The red cabbage extract did not change the total Pb uptake but it enhanced the translocation of accumulated metal from roots to shoots. Within the pretreated roots, more Pb was deposited in their basal part and definitely smaller amount of the metal was bound in the apoplast of the outer layers of cortex cells. The ultrastructural analysis (transmission electron microscopy and X-ray microanalysis) revealed that the ATH-rich extract lowered the number of Pb deposits in intracellular spaces, cell wall and cytoplasm of root meristematic cells as well as in such organelles important to cell metabolism as mitochondria, plastids and nucleus. The Pb deposits were preferably localised in those vacuoles where ATH also occurred. This sequestration of Pb in vacuoles is probably responsible for reduction of metal cytotoxicity and consequently could lead to better plant growth.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa.

BACKGROUND AND AIMS: Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. METHODS: Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H(2)O(2) production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). KEY RESULTS: Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H(2)O(2), γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. CONCLUSIONS: The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.

Biophysical methods for assessing plant responses to nanoparticle exposure.

As nanotechnology rapidly emerges into a new industry-driven by its enormous potential to revolutionize electronics, materials, and medicine-exposure of living species to discharged nanoparticles has become inevitable. Despite the increased effort on elucidating the environmental impact of nanotechnology, literature on higher plants exposure to nanoparticles remains scarce and often contradictory. Here we present our biophysical methodologies for the study of carbon nanoparticle uptake by Allium cepa cells and rice plants. We address the three essential aspects for such studies: identification of carbon nanoparticles in the plant species, quantification of nanotransport and aggregation in the plant compartments, and evaluation of plant responses to nanoparticle exposure on the cellular and organism level. Considering the close connection between plant and mammalian species in ecological systems especially in the food chain, we draw a direct comparison on the uptake of carbon nanoparticles in plant and mammalian cells. In addition to the above studies, we present methods for assessing the effects of quantum dot adsorption on algal photosynthesis.

The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins.

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei.

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector.

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.

Cellular force microscopy for in vivo measurements of plant tissue mechanics.

Although growth and morphogenesis are controlled by genetics, physical shape change in plant tissue results from a balance between cell wall loosening and intracellular pressure. Despite recent work demonstrating a role for mechanical signals in morphogenesis, precise measurement of mechanical properties at the individual cell level remains a technical challenge. To address this challenge, we have developed cellular force microscopy (CFM), which combines the versatility of classical microindentation techniques with the high automation and resolution approaching that of atomic force microscopy. CFM's large range of forces provides the possibility to map the apparent stiffness of both plasmolyzed and turgid tissue as well as to perform micropuncture of cells using very high stresses. CFM experiments reveal that, within a tissue, local stiffness measurements can vary with the level of turgor pressure in an unexpected way. Altogether, our results highlight the importance of detailed physically based simulations for the interpretation of microindentation results. CFM's ability to be used both to assess and manipulate tissue mechanics makes it a method of choice to unravel the feedbacks between mechanics, genetics, and morphogenesis.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols.

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ~1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ(2)=1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

Functional characterization of an apple apomixis-related MhFIE gene in reproduction development.

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple.

High-resolution full-field optical coherence microscopy using a Mirau interferometer for the quantitative imaging of biological cells.

In this paper quantitative imaging of biological cells using high-resolution full-field optical coherence microscopy (FF-OCM) is reported. The FF-OCM was realized using a swept-source system, a Mirau interferometer, and a CCD camera (a two-dimensional detection unit). A Mirau-interferometric objective lens was used to generate the interferometric signal. The signal was analyzed by a Fourier analysis technique. Optically sectioned amplitude images and a quantitative phase map of biological cells such as onion skin and red blood cells (RBCs) are demonstrated. Further, the refractive index profile of the RBCs is also presented. For the 50× Mirau objective, the experimentally achieved axial and transverse resolution of the present system are 3.8 and 1.2 µm, respectively. The CCD provides parallel detection and measures enface images without X, Y, Z mechanical scanning.

Evaluation of cytogenotoxic effects of cold aqueous extract from Achyrocline satureioides by Allium cepa L test.

Achyrocline satureioides ("marcela del campo") is native to America. Numerous investigations have reported several bioactive properties such as anti-inflammatory, hepatoprotective, immunomodulatory, antimicrobial and antiviral. Nowadays, few medicinal plants have been scientifically evaluated to test its safety, efficacy and potential benefits, despite the great public interest in these herbs. The aim of this work was to evaluate the cytotoxic and genotoxic activities of cold aqueous extract obtained from A. satureioides using Allium cepa L test. The results demonstrated the absence of genotoxicity of the extract. Only higher concentrations induced cytotoxicity but interestingly this effect was reversible and was not associated with mutagenicity. The contribution of this research provides assurance of safety in the application of Achyrocline satureioides in treatment of microbial diseases and other pathologies helping to define selective toxicity.