Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Mg2+ Is a Missing Link in Plant Cell Ca2+ Signalling and Homeostasis-A Study on Vicia faba Guard Cells.

Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types, where they have an important role in Ca2+-dependent signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca2+ into cells. HACCs are permeable to Ca2+, Ba2+, and Mg2+; activated by H2O2 and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca2+ ([Ca2+]Cyt), and hence closes during [Ca2+]Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ -200 mV, and that Mg2+ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg2+ levels affect HACC gating and that channel opening requires either high negative voltages (≥ -200 mV) or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCs described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved diacidic Mg2+ binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

Malathion and dithane induce DNA damage in Vicia faba.

The increasing use of pesticides such as malathion and dithane in agriculture causes environmental mutagenicity. However, their genotoxicity in edible crops is seldom assessed. In this study, the genotoxic potential of malathion and dithane was evaluated in the roots of Vicia faba L. All three concentrations (0.05, 0.1, and 0.2%) of malathion and dithane tested resulted in a significant decrease in root length and inhibited seed germination. Cytological observations showed that the mitotic frequency in the root meristematic cells decreased parallel to the increase in concentrations, and the increase in chromosome aberrations and micronuclei frequency was concentration dependent. Alkaline comet assay revealed significant onset of DNA damage at all tested concentrations. For the randomly amplified polymorphic (RAPD)-polymerase chain reaction (PCR) analyses, 10 random RAPD primers were found to produce 116 unique polymorphic RAPD band fragments of 223-3139 bp. Each primer generated 3-15 RAPD bands on an average. The percentage of polymorphic DNA fragments was higher in malathion-exposed plants than dithane ones. The changes in RAPD profiles included disappearance and/or appearance of DNA bands in malathion and dithane treatment. Hence, DNA damage observed by the cytogenetic endpoints and comet assay corroborated with RAPD-PCR analysis. A total of 15 new protein bands of molecular weight ranging 11.894-226.669 kDa were observed in roots of Vicia plants that were exposed to the pesticides. The number of new protein bands was higher in malathion-treated DNA samples than in dithane-treated ones. Based on the results, we conclude that the pesticides can alter genomic template stability and change protein profiles. Malathion was more genotoxic than dithane. Therefore, RAPD assays can be useful in determining genotoxicity of pesticides in V. faba and other crops along with other quantitative parameters.

Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require phosphatidylinositol 3,5-bisphosphate.

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution.

Live Single-Cell Plant Hormone Analysis by Video-Mass Spectrometry.

Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone-mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of ABA and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem mass spectrometry (MS/MS) analysis. Stable isotope-labeled [D(6)]ABA or [(13)C(6)]JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single-cell analysis were compared with levels found by analysis of purified extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells.

Plasma Membrane Ca2+-Permeable Channels are Differentially Regulated by Ethylene and Hydrogen Peroxide to Generate Persistent Plumes of Elevated Cytosolic Ca2+ During Transfer Cell Trans-Differentiation.

The enhanced transport capability of transfer cells (TCs) arises from their ingrowth wall architecture comprised of a uniform wall on which wall ingrowths are deposited. The wall ingrowth papillae provide scaffolds to amplify plasma membranes that are enriched in nutrient transporters. Using Vicia faba cotyledons, whose adaxial epidermal cells spontaneously and rapidly (hours) undergo a synchronous TC trans-differentiation upon transfer to culture, has led to the discovery of a cascade of inductive signals orchestrating deposition of ingrowth wall papillae. Auxin-induced ethylene biosynthesis initiates the cascade. This in turn drives a burst in extracellular H2O2 production that triggers uniform wall deposition. Thereafter, a persistent and elevated cytosolic Ca(2+) concentration, resulting from Ca(2+) influx through plasma membrane Ca(2+)-permeable channels, generates a Ca(2+) signal that directs formation of wall ingrowth papillae to specific loci. We now report how these Ca(2+)-permeable channels are regulated using the proportionate responses in cytosolic Ca(2+) concentration as a proxy measure of their transport activity. Culturing cotyledons on various combinations of pharmacological agents allowed the regulatory influence of each upstream signal on Ca(2+) channel activity to be evaluated. The findings demonstrated that Ca(2+)-permeable channel activity was insensitive to auxin, but up-regulated by ethylene through two independent routes. In one route ethylene acts directly on Ca(2+)-permeable channel activity at the transcriptional and post-translational levels, through an ethylene receptor-dependent pathway. The other route is mediated by an ethylene-induced production of extracellular H2O2 which then acts translationally and post-translationally to up-regulate Ca(2+)-permeable channel activity. A model describing the differential regulation of Ca(2+)-permeable channel activity is presented.

Differential transcriptional networks associated with key phases of ingrowth wall construction in trans-differentiating epidermal transfer cells of Vicia faba cotyledons.

BACKGROUND: Transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. The ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. However, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood. RESULTS: An RNAseq study of an inducible epidermal transfer cell system in cultured Vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. All functional groups of genes examined were expressed before and following transition to a transfer cell fate. What changed were the isoform profiles of expressed genes within functional groups. Genes encoding ethylene and Ca(2+) signal generation and transduction pathways were enriched during uniform wall construction. Auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and ABA genes were evenly expressed across ingrowth wall construction. Expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. Uniform wall and wall ingrowth specific expression of exocyst complex components and SNAREs suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. Key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. Transfer cell specific expression of cellulose synthases was absent. Rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. More striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. Extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. Ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of ABC transporters. Sugar transporters were less represented. CONCLUSIONS: Pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. Each phase of ingrowth wall construction was linked with unique cohorts of expressed genes.

Contributions of photosynthetic and non-photosynthetic cell types to leaf respiration in Vicia faba†L. and their responses to growth temperature.

In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed.

Polarized and persistent Ca²âº plumes define loci for formation of wall ingrowth papillae in transfer cells.

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited.

A novel structural effector from rust fungi is capable of fibril formation.

It has been reported that filament-forming surface proteins such as hydrophobins are important virulence determinants in fungi and are secreted during pathogenesis. Such proteins have not yet been identified in obligate biotrophic pathogens such as rust fungi. Rust transferred protein 1 (RTP1p), a rust protein that is transferred into the host cytoplasm, accumulates around the haustorial complex. To investigate RTP1p structure and function, we used immunocytological, biochemical and computational approaches. We found that RTP1p accumulates in protuberances of the extra-haustorial matrix, a compartment that surrounds the haustorium and is separated from the plant cytoplasm by a modified host plasma membrane. Our analyses show that RTP1p is capable of forming filamentous structures in vitro and in vivo. We present evidence that filament formation is due to ß-aggregation similar to what has been observed for amyloid-like proteins. Our findings reveal that RTP1p is a member of a new class of structural effectors. We hypothesize that RTP1p is transferred into the host to stabilize the host cell and protect the haustorium from degradation in later stages of the interaction. Thus, we provide evidence for transfer of an amyloid-like protein into the host cell, which has potential for the development of new resistance mechanisms against rust fungi.

The crucial elements of the 'last step' of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings.

KEY MESSAGE: Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process. The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4',6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A3 (CMA3)-stained chromocenters, (3) increase in fluorescence intensity of CMA3-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA3-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn(2+) and Ca(2+)/Mg(2+)-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification "in Byczkowska et al. (Protoplasma 250:121-128, 2013)"], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.

Exogenous application of salicylic acid to alleviate the toxic effects of insecticides in Vicia faba L.

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from insecticides toxicity. The seeds of Vicia faba var IIVR Selection-1 were treated with different concentrations (1.5, 3.0, and 6.0 ppm) of the insecticides alphamethrin (AM) and endosulfan (ES) for 6 h with and without 12 h conditioning treatment of SA (0.01 mM). Insecticides treatment caused a significant decrease in mitotic index (MI) and induction of different types of chromosomal abnormalities in the meristematic cells of broad bean roots. Pretreatment of seeds with SA resulted in increased MI and significant reduction of chromosomal abnormalities. SA application also regulated proline accumulation and carotenoid content in the leaf tissues. SA resulted in the decrement of insecticides induced increase in proline content and increased the carotenoids content. These results illustrate the ameliorating effect of SA under stress conditions and reveal that SA is more effective in alleviating the toxic effects of insecticides at higher concentrations than that at lower concentrations.

Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons.

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.

Anion channel sensitivity to cytosolic organic acids implicates a central role for oxaloacetate in integrating ion flux with metabolism in stomatal guard cells.

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily Mal (malate), and its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (I(Cl)) by cytosolic Mal or its immediate metabolite OAA (oxaloacetate). In the present study, we have examined the impact of Mal, OAA and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect I(Cl), but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of ICl by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to co-ordinate organic acid metabolism with I(Cl) through the direct effect of organic acid pool size. The findings of the present study also add perspective to in vivo recordings using acetate-based electrolytes.

Lead-induced genotoxicity to Vicia faba L. roots in relation with metal cell uptake and initial speciation.

Formation of organometallic complexes in soil solution strongly influence metals phytoavailability. However, only few studies deal with the influence of metal speciation both on plant uptake and genotoxicity. In the present study, Vicia faba seedlings were exposed for 6h in controlled hydroponic conditions to 5 µM of lead nitrate alone and chelated to varying degrees by different organic ligands. Ethylenediaminetetraacetic acid and citric acid were, respectively, chosen as models of humic substances and low weight organic acids present in natural soil solutions. Visual Minteq software was used to estimate free lead cations concentration and ultimately to design the experimental layout. For all experimental conditions, both micronucleus test and measure of lead uptake by plants were finally performed. Chelation of Pb by EDTA, a strong chelator, dose-dependently increased the uptake in V. faba roots while its genotoxicity was significantly reduced, suggesting a protective role of EDTA. A weak correlation was observed between total lead concentration absorbed by roots and genotoxicity (r(2)=0.65). In contrast, a strong relationship (r(2)=0.93) exists between Pb(2+) concentration in exposure media and genotoxicity in the experiment performed with EDTA. Citric acid induced labile organometallic complexes did not demonstrate any significant changes in lead genotoxicity or uptake. These results demonstrate that metal speciation knowledge could improve the interpretation of V. faba genotoxicity test performed to test soil quality.

Mechanism of kinetin-induced death of Vicia faba ssp. minor root cortex cells.

Cell death (CD) may be induced by endogenous or exogenous factors and contributes to all the steps of plant development. This paper presents results related to the mechanism of CD regulation induced by kinetin (Kin) in the root cortex of Vicia faba ssp. minor. To explain the process, 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55), adenine (Ad), 5'-amine-5'-deoxyadenosine (Ado) and N-(2-chloro-4-piridylo)-N'-phenylurea (CPPU) were applied to (i) block cytokinin receptors (CKs) and inhibit the activities of enzymes of CK metabolism, i.e., (ii) phosphoribosyltransferase, (iii) kinases, and (iv) oxidases, respectively. Moreover, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), lanthanum chloride (LaCl3), ruthenium red (RRed) and cyclosporine A (CS-A) were applied to (i) chelate extracellular calcium ions (Ca2+) as well as blocks of (ii) plasma-, (iii) endoplasmic reticulum- (ER) membrane Ca2+ ion channels and (iv) mitochondria- (MIT) Ca2+ ions release by permeability transition por (PTP), respectively. The measured physiological effectiveness of these factors was the number of living and dying cortex cells estimated with orange acridine (OA) and ethidium bromide (EB), the amounts of cytosolic Ca2+ ions with chlortetracycline (CTC) staining and the intensity of chromatin and Ca2+-CTC complex fluorescence, respectively. Moreover, the role of sorafenib, an inhibitor of RAF kinase, on the vitality of cortex cells and ethylene levels as well as the activities of RAF-like kinase and MEK2 with Syntide-2 and Mek2 as substrates were studied. The results clarified the previously presented suggestion that Kin is converted to appropriate ribotides (5'-monophosphate ribonucleotides), which cooperate with the ethylene and Ca2+ ion signalling pathways to transduce the signal of kinetin-programmed cell death (Kin-PCD). Based on the present and previously published results related to Kin-PCD, the crosstalk between ethylene and MAP kinase signalling, as well as inhibitors of CK receptors and enzymes of their metabolism, is proposed.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions.

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

Hydrogen sulphide, a novel gasotransmitter involved in guard cell signalling.

Hydrogen sulphide (H(2) S) has been proposed as the third gasotransmitter. In animal cells, H(2) S has been implicated in several physiological processes. H(2) S is endogenously synthesized in both animals and plants by enzymes with l-Cys desulphydrase activity in the conversion of l-Cys to H(2) S, pyruvate and ammonia. The participation of H(2) S in both stomatal movement regulation and abscisic acid (ABA)-dependent induction of stomatal closure was studied in epidermal strips of three plant species (Vicia faba, Arabidopsis thaliana and Impatiens walleriana). The effect of H(2) S on stomatal movement was contrasted with leaf relative water content (RWC) measurements of whole plants subjected to water stress. In this work we report that exogenous H(2) S induces stomatal closure and this effect is impaired by the ATP-binding cassette (ABC) transporter inhibitor glibenclamide; scavenging H(2) S or inhibition of the enzyme responsible for endogenous H(2) S synthesis partially blocks ABA-dependent stomatal closure; and H(2) S treatment increases RWC and protects plants against drought stress. Our results indicate that H(2) S induces stomatal closure and participates in ABA-dependent signalling, possibly through the regulation of ABC transporters in guard cells.