[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.

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.

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility.

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.

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.

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.

High resolution scanning electron microscopy of plasmodesmata.

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.

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.

The effects of the anthocyanin-rich extract from red cabbage leaves on Allium cepa L. root tip cell ultrastructure.

The effect of exogenously applied 250 µM anthocyanin-rich (ATH-rich) extract from red cabbage leaves on the ultrastructure of Allium cepa root meristematic cells was investigated. The tested extract slightly affected mitochondria, endoplasmic reticulum (ER), Golgi apparatus and vacuoles. In the presence of ATH, 62% of mitochondria converted to condensed type. In addition swollen, circular ER cisternae were sporadically observed. In the ATH-treated roots, one third of Golgi structures was characterized by the reduced number of vesicles. Moreover in 54% of vacuoles, the electron-dense granular and circular material appeared. Additionally, in the cytoplasm, the presence of numerous multivesicular bodies (MVB) was noticed. The observed ultrastructural modifications of mitochondria, and presumably also ER, probably resulted from the ability of an ATH to affect mitochondrial respiratory activity. The other changes in A. cepa root meristematic cell ultrastructure were connected with the transport of exogenously applied ATH into vacuoles. It seems that they are transported from the plasmolemma to the vacuole by multivesicular bodies (MVB), and there trapped by anthocyanic vacuolar inclusions (AVIs). However, none of the observed ultrastructural changes seemed to disturb cell functions, therefore the ATH-rich extract from red cabbage leaves may be regarded as cell-friendly and can be safely used as a detoxifying agent against heavy metal poisoning, as it is more and more often postulated.

Spent Pot Liner (SPL) induced DNA damage and nuclear alterations in root tip cells of Allium cepa as a consequence of programmed cell death.

There are various toxic effects of environmental pollutants, including apoptosis and carcinogenesis. Spent Pot Liner (SPL) is solid waste from the aluminum industry. It has a highly variable composition, including cyanide, fluoride, organics and metals. Preliminary characterizations of the effect of SPL on Allium cepa show the presence of condensed nuclei. Thus, the aim of this study was to analyze the toxic effect of SPL in A. cepa root meristem in the context of programmed cell death (PCD). A lot of specific features of this process such as DNA fragmentation, condensed chromatin, spherical nuclei and the formation of apoptotic-like bodies were observed in root meristem after SPL treatment. Root meristem treated with SPL 25% solution exhibited an alteration in antioxidant enzyme activities; a reduction in NCR as a consequence of high percentage of condensed nuclei; DNA fragmentation, detected by electrophoresis and TUNEL assay; cytoplasm vacuolization and also a disturbance in root morphology. These features are associated with programmed cell death (PCD) under abiotic stress. Therefore, these data show that SPL induces apoptosis-like PCD in root meristem cells of A. cepa.

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.

Subnuclear compartmentalization and function of actin and nuclear myosin I in plants.

Actins are highly conserved proteins that serve as the basic building blocks of cytoskeletal microfilaments. In animal cells, specific nuclear actin adopts unconventional conformations that are involved in multiple nuclear functions and that associate with nuclear actin binding proteins. However, there is practically no information available about nuclear actin in plants. Indeed, actin has not been detected in the nuclear proteomes of many plants, and orthologs of the main structural nuclear actin-binding proteins have yet to be identified. Here, we have investigated the characteristics, intranuclear compartmentalization, and function of actin in isolated Allium cepa nuclei as well as that of its motor protein nuclear myosin I (NMI). Using conformation-specific antibodies for nuclear actin isoforms, ss-actin, and NMI, the distribution of these proteins was studied in Western blots and by immunocytochemistry. Moreover, the participation of nuclear actin in transcription was analyzed in run on in situ assays and inhibition of RNA polymerases I and II. We show that actin isoforms with distinct solubilities are present in onion nuclei with a consistent subnuclear compartmentalization. Actin and NMI are highly enriched in foci that are similar to transcription foci, although actin is also distributed diffusely in the nucleus and nucleolus as well as accumulating in a subset of the Cajal bodies. Immunogold labeling identified both proteins in the nuclear transcription subdomains and in other subnuclear compartments. In addition, actin and NMI were diffusely distributed in the nuclear matrix.

[Antimitotic activity of new 2,6-dinitroaniline derivatives and their synergistic activity in compositions with graminicides].

Shown antimicrotrubules activity of new 2,6-dinitroaniline compounds. Investigated their ability on apoptotic processes in a plant cell when used as a composition with inhibitors of acetyl-CoA carboxylase.

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.

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

[Screening of new 2,4- and 2,6-dinitroaniline derivates for phytotoxicity and antimitotic activity].

The results of Allium-test screening of new 2,4- and 2,6-dinitroaniline derivates on antimitotic activity and phytotoxicity are presented in the work. It is revealed that all studied compounds which are derivates of 2,4-dinitroaniline, 2,6-dinitro-(4-fluoromethyl)-aniline as well as (methylsulfonyl) nitrobenzol, can evoke a change in mitotic index value, an appearance of cytogenetic damages and also have phytotoxic effect on Allium cepa seedling roots. On data obtained the continuation of investigation the action of N-(2,4-dinitrophenyl)-orto-aminobenzoic acid, N,N-diethyl-2,6-dinitro-4-(trifluoromethyl)-aniline and 1-methylsulfonyl-3-nitrobenzol as potential herbicides is proposed.

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.

A new bio-inspired route to metal-nanoparticle-based heterogeneous catalysts.

Onion-type multilamellar vesicles are made of concentric bilayers of organic surfactant and are mainly known for their potential applications in biotechnology. They can be used as microreactors for the spontaneous and controlled production of metal nanoparticles. This process does not require any thermal treatment and, hence, it is also attractive for material sciences such as heterogeneous catalysis. In this paper, silver-nanoparticle-based catalysts are prepared by transferring onion-grown silver nanoparticles onto inorganic supports. The resulting materials are active in the total oxidation of benzene, attesting that this novel bio-inspired concept is promising in inorganic catalysis.