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.

An ethylene biosynthesis enzyme controls quantitative variation in maize ear length and kernel yield.

Maize ear size and kernel number differ among lines, however, little is known about the molecular basis of ear length and its impact on kernel number. Here, we characterize a quantitative trait locus, qEL7, to identify a maize gene controlling ear length, flower number and fertility. qEL7 encodes 1-aminocyclopropane-1- carboxylate oxidase2 (ACO2), a gene that functions in the final step of ethylene biosynthesis and is expressed in specific domains in developing inflorescences. Confirmation of qEL7 by gene editing of ZmACO2 leads to a reduction in ethylene production in developing ears, and promotes meristem and flower development, resulting in a ~13.4% increase in grain yield per ear in hybrids lines. Our findings suggest that ethylene serves as a key signal in inflorescence development, affecting spikelet number, floral fertility, ear length and kernel number, and also provide a tool to improve grain productivity by optimizing ethylene levels in maize or in other cereals.

Quantitative cell biology of tip growth in moss.

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.

WUSCHEL triggers innate antiviral immunity in plant stem cells.

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.

The Cyclin CYCA3;4 Is a Postprophase Target of the APC/CCCS52A2 E3-Ligase Controlling Formative Cell Divisions in Arabidopsis.

The anaphase promoting complex/cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CELL DIVISION PROTEIN20 (CDC20) and CELL CYCLE SWITCH52 (CCS52). Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis (Arabidopsis thaliana) CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhance the scored ccs52a2-1 phenotypes. Furthermore, whereas the CYCA3;4 protein is promptly broken down after prophase in wild-type plants, it remains present in later stages of mitosis in ccs52a2-1 mutant plants, marking it as a putative APC/CCCS52A2 substrate. Strikingly, increased CYCA3;4 levels result in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4 gain-of-function plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 contributes to disorganized formative divisions, possibly in part caused by the inactivation of RBR1.

Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem.

RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) 'Moneymaker' tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in 'Moneymaker' tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa.

KEY MESSAGE: Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Function of NHX-type transporters in improving rice tolerance to aluminum stress and soil acidity.

MAIN CONCLUSION: In this study, we show that ectopic expression of either HtNHX1 or HtNHX2, from Helianthus tuberosus plant (located at vacuolar and endosome membranes, respectively), in rice plants could enhance its tolerance to aluminum (Al3+) stress and soil acidity. Plant sodium (potassium)/proton (Na+(K+)/H+ antiporters of the NHX family have been extensively characterized as they are related to the enhancement of salt tolerance. However, no previous study has reported NHX transporter functions in plant tolerance to Al3+ toxicity. In this study, we demonstrate their role as a component of the Al3+ stress tolerance mechanism. We show that the ectopic expression of either HtNHX1 or HtNHX2 , from Helianthus tuberosus plant, in rice (located at vacuole and endosome, respectively) could also enhance rice tolerance to Al3+ stress and soil acidity. Expression of either HtNHX1 or HtNHX2 reduced the inhibitory effect of Al3+ on the rice root elongation rate; both genes were reported to be equally effective in improvement of stress conditions. Expression of HtNHX1 enhanced Al3+-trigged-secretion of citrate acids, rhizosphere acidification, and also reduced K+ efflux from root tissues. In contrast, expression of HtNHX2 prevented Al3+-trigged-decrease of H+ influx into root tissues. Al3+-induced damage of the cell wall extensibility at the root tips was impaired by either HtNHX1 or HtNHX2. Co-expression of HtNHX1 and HtNHX2 further improved rice growth, particularly under the Al3+ stress conditions. The results demonstrate that HtNHX1 and HtNHX2 improved rice tolerance to Al3+ via different mechanisms by altering the K+ and H+ fluxes and the cell wall structure.

Cell Size Control in Plants.

The genetic control of the characteristic cell sizes of different species and tissues is a long-standing enigma. Plants are convenient for studying this question in a multicellular context, as their cells do not move and are easily tracked and measured from organ initiation in the meristems to subsequent morphogenesis and differentiation. In this article, we discuss cell size control in plants compared with other organisms. As seen from yeast cells to mammalian cells, size homeostasis is maintained cell autonomously in the shoot meristem. In developing organs, vacuolization contributes to cell size heterogeneity and may resolve conflicts between growth control at the cellular and organ levels. Molecular mechanisms for cell size control have implications for how cell size responds to changes in ploidy, which are particularly important in plant development and evolution. We also discuss comparatively the functional consequences of cell size and their potential repercussions at higher scales, including genome evolution.

The induction and maintenance of in vitro plant morphogenesis as viewed from a new perspective, with theoretical and constructive implications.

In plant tissue culture research, the non-traditional growth regulators, methylglyoxal and ascorbic acid, have been used to induce and promote in vitro morphogenesis from plant callus, generally having the initial characteristics of a type of neoplasm, and in many cases overcoming recalcitrant morphogenesis. In other investigations methylglyoxal, most likely with ascorbic acid, also promoted such morphogenesis. In the various investigations, low concentrations of methylglyoxal were used and proved to be the most effective in promoting in vitro morphogenesis. In many cases, the growth of such neoplastic-like calli was concurrently inhibited on culture media containing these chemicals. When methylglyoxal was present in high concentration, morphogenesis was also inhibited. Such chemicals, it would appear likely, allowed for the generation of cohesive forces within regions of the calli, reversing the neoplastic state in such regions, due to very low internal cohesion, and through such cohesive forces of particular magnitude, morphogenesis ensued, as an adaptive response to the stress of such cohesive forces. This would suggest a deeper, underlying biological process, with developmental features, that is perhaps universal among plants and perhaps in all biological organisms. This particular, consistent avenue and theme of plant tissue culture research, manifested over four decades and across four continents, may have revealed a unifying, dynamical process in both the biological and physical worlds, with constructive implications for agriculture and medicine.

Toxicological findings about an anticancer fraction with casearins described by traditional and alternative techniques as support to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts, essential oils and molecules from Casearia sylvestris have popularly shown pharmacological actions against chronic diseases, as anxiety, inflammation, cancer and dyslipidemia. In the context of antitumoral therapy, we investigated in vitro, ex vivo and in vivo toxicological changes induced by a Fraction with Casearins (FC) and its component Casearin X isolated from C. sylvestris on animal and vegetal cells, and upon invertebrates and mammals. MATERIAL AND METHODS: Cytotoxicity was carried out using normal lines and absorbance and flow cytometry techniques, Artemia salina nauplii, Danio rerio embryos and meristematic cells from Allium cepa roots. Acute and 30 days-mice analysis were done by behavioral, hematological and histological investigations and DNA/chromosomal damages detected by alkaline Cometa and micronucleus assays. RESULTS: FC was cytotoxic against lung and fibroblasts cells and caused DNA breaks, loss of integrity and mitochondrial depolarization on ex vivo human leukocytes. It revealed 24 h-LC50 values of 48.8 and 36.7 µg/mL on A. salina nauplii and D. rerio embryos, reduced mitotic index of A. cepa roots, leading to cell cycle arrest at metaphase and anaphase and micronuclei. FC showed i.p. and oral LD50 values of 80.9 and 267.1 mg/kg body weight. Subacute i.p. injections induced loss of weight, swelling of hepatocytes and tubules, tubular and glomerular hemorrhage, microvesicular steatosis, lung inflammatory infiltration, augment of GPT, decrease of albumin, alkaline phosphatase, glucose, erythrocytes, and lymphocytes, and neutrophilia (p > 0.05). FC-treated animals at 10 mg/kg/day i.p. caused micronuclei in bone marrow and DNA strand breaks in peripheral leukocytes. CONCLUSIONS: This research postulated suggestive side effects after use of FC-related drugs, demonstrating FC as antiproliferative and genotoxic on mammal and meristematic cells, including human leukocytes, teratogenicity upon zebrafish embryos, myelosuppression, clastogenicity, and morphological and biochemical markers indicating liver as main target for FC-induced systemic toxicity.

Root stem cell niche maintenance and apical meristem activity critically depend on THREONINE SYNTHASE1.

Indeterminate root growth depends on the stem cell niche (SCN) and root apical meristem (RAM) maintenance whose regulation permits plasticity in root system formation. Using a forward genetics approach, we isolated the moots koom1 ('short root' in Mayan) mutant that shows complete primary RAM exhaustion and abolished SCN activity. We identified that this phenotype is caused by a point mutation in the METHIONINE OVERACCUMULATOR2 (MTO2) gene that encodes THREONINE SYNTHASE1 and renamed the mutant as mto2-2. The amino acid profile showed drastic changes, most notorious of which was accumulation of methionine. In non-allelic mto1-1 (Arabidopsis thaliana cystathionine gamma-synthetase1) and mto3-1 (S-adenosylmethionine synthetase) mutants, both with an increased methionine level, the RAM size was similar to that of the wild type, suggesting that methionine overaccumulation itself did not cause RAM exhaustion in mto2 mutants. When mto2-2 RAM is not yet completely exhausted, exogenous threonine induced de novo SCN establishment and root growth recovery. The threonine-dependent RAM re-establishment in mto2-2 suggests that threonine is a limiting factor for RAM maintenance. In the root, MTO2 was predominantly expressed in the RAM. The essential role of threonine in mouse embryonic stem cells and in RAM maintenance suggests that common regulatory mechanisms may operate in plant and animal SCN maintenance.

Comparative physiological analysis in the tolerance to salinity and drought individual and combination in two cotton genotypes with contrasting salt tolerance.

Soil salinity and drought are the two most common and frequently co-occurring abiotic stresses limiting cotton growth and productivity. However, physiological mechanisms of tolerance to such condition remain elusive. Greenhouse pot experiments were performed to study genotypic differences in response to single drought (4% soil moisture; D) and salinity (200 mM NaCl; S) stress and combined stresses (D + S) using two cotton genotypes Zhongmian 23 (salt-tolerant) and Zhongmian 41 (salt-sensitive). Our results showed that drought and salinity stresses, alone or in combination, caused significant reduction in plant growth, chlorophyll content and photosynthesis in the two cotton genotypes, with the largest impact visible under combined stress. Interestingly, Zhongmian 23 was more tolerant than Zhongmian 41 under the three stresses and displayed higher plant dry weight, photosynthesis and antioxidant enzymes activities such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX) activities compared to control, while those parameters were significantly decreased in salt-stresses Zhongmian 41 compared to control. Moreover, Na+ /K+ -ATPase activity was more enhanced in Zhongmian 23 than in Zhongmian 41 under salinity stress. However, under single drought stress and D + S stress no significant differences were observed between the two genotypes. No significant differences were detected in Ca2+ /Mg2+ -ATPase activity in Zhongmian 41, while in Zhongmian 23 it was increased under salinity stress. Furthermore, Zhongmian 23 accumulated more soluble sugar, glycine-betaine and K+ , but less Na+ under the three stresses compared with Zhongmian 41. Obvious changes in leaf and root tips cell ultrastructure was observed in the two cotton genotypes. However, Zhongmian 23 was less affected than Zhongmian 41 especially under salinity stress. These results give a novel insight into the mechanisms of single and combined effects of drought and salinity stresses on cotton genotypes.

Influence of seed priming with iron and/or zinc in the nucleolar activity and protein content of bread wheat.

Seed priming with iron (Fe) and/or zinc (Zn) can overcome the reduced availability of these micronutrients in soils and crops, but suitable dosages should be predetermined. Nucleolus responds to stress, such as cytotoxicity, with alterations perceivable by cytogenetic analyses. This work intends to study how seed priming with Fe and/or Zn affects the nucleolar activity in roots and the total soluble protein content in the flour of bread wheat cv. 'Jordão'. Seven priming treatments with 0 mg L-1 to 8 mg L-1 of Fe and/or Zn were performed. In all treatments, each metaphase cell presented a maximum of six nucleolar organizer regions positively stained with silver nitrate (Ag-NORs). Also, a maximum number of six nucleoli per nucleus were observed in all treatments, except in the hydroprimed seeds (used as control) that showed a maximum of five nucleoli, probably due to nucleolar fusion. Irregular interphases were frequent in treatments with the highest dosage of micronutrients (8 mg L-1 Fe and/or 8 mg L-1 Zn). The nucleolar area reduced (p < 0.001) as the number of nucleoli increased, and it was lower in treatments with a combination of Fe and Zn. However, the combinations of Fe and Zn showed the highest concentrations of total soluble protein (p ≤ 0.001). Although a reduced nucleolar area represents low ribosomal RNA gene transcription and ribosomal production, the significant increase of the number of nucleoli in the seeds primed with Fe and Zn enhanced the total soluble protein content as compared to the hydroprimed seeds (control) probably due to an increase of nucleolar surface-to-volume ratio that improved the protein synthesis. Overall, this work revealed that priming of bread wheat seeds with suited dosages of Fe and Zn can improve the nutritional value of flour, and the nucleolar number, morphology, and area can be useful biomarkers in cytotoxicity studies.

Phytosynthesis of gold and silver nanoparticles enhance in vitro antioxidant and mitostimulatory activity of Aconitum toxicum Reichenb. rhizomes alcoholic extracts.

Extracts obtained from different plant species proved to be a valuable tool in various biomedical applications. In the same time, the phytosynthesis of noble metal nanoparticles represents an already well-established route for obtaining nanoparticles with biological activity. The present paper studies the antioxidant activity and the cytogenetic effects of the alcoholic extracts from rhizomes of Aconitum toxicum Rchb., before and after the phytosynthesis of gold and silver nanoparticles, on the meristematic root cells of Allium cepa L., and on the general mitotic index and the progression rate through the mitotic phases, respectively, as well as on the genetic material organized in chromosomes. The extracts were characterized in terms of total polyphenolics content (1.49% and, respectively, 2.29%) and aconitine content (by HPLC - 4.891 mg/L and, respectively, 18.211 mg/L), while the phytosynthesis of metallic nanoparticles was monitored by UV-Vis spectrometry, X-ray diffraction, X-ray fluorescence and electron microscopy. Both the extracts and the obtained nanoparticles were evaluated for antioxidant potential (the antioxidant activity ranging between 78% and 84.32%) and cytogenetic effects. The obtained results prove the phytosynthesis of AgNPs and AuNPs with dimensions ranging from 9 nm to 15 nm for AuNPs, respectively from 53 nm to 67 nm for AgNPs. The extracts obtained from rhizomes of A. toxicum Rchb. induced mitotic stress, as well as a series of nuclear and mitotic aberrations. The biosynthesis of AgNPs and AuNPs intensified the antioxidant and mitostimulatory activity of the extracts.

Cytogenetic and genotoxic effects of Rosmaniric Acid on Allium cepa L. root meristem cells.

Rosmarinic acid (RA) is a natural polyphenol carboxylic acid, an ester of caffeic acid with 3,4-dihydroxyphenyllactic acid, found in many species. Current study was aimed to investigate the mitotic division, chromosomal and genotoxic effects of RA on Allium cepa root meristematic cells. In Allium root growth inhibition test, EC50 value was found as 100 ppm. Three concentrations (50, 100, and 200 ppm) of RA under different exposure periods (24, 48, 72 and 96 h) were employed to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 ppm) were used as a negative and positive control, respectively. 100 (except 24 h) and 200 ppm of RA significantly decreased mitotic index (MI). There was an increase of total chromosomal aberrations (CAs) at 50 ppm and simultaneous decrease of CAs at 200 ppm concentrations (p < 0.05). A significant increase in DNA damage was also observed at 200 ppm by Comet assay. Quantitative analysis of RA in A. cepa root meristem cells was also done by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Further investigations are required to explore the molecular mechanism involved in the cytotoxicity and genotoxicity of RA on plants.

Toxic, cytotoxic and genotoxic potential of synthetics food flavorings / Potencial tóxico, citotóxico y genotóxico de saborizantes sintéticos de alimentos

Food flavorings in general are few studied for the toxicological aspect. This condition justifies toxicity, cytotoxicity and genotoxicity assessments of the substances. In the present study, the toxicity of banana, cherry and hazelnut flavorings was evaluated in meristematic cells of roots of Allium cepa, in pure form (as marketed) and in the concentrations of 12.5; 25 and 50%, after 24 and 48 hours of exposure. Toxic potential of these food additives was also evaluated against Artemia salina nauplii at concentrations of 0.78; 1.56; 3.12; 6.25; 12.5; 25 and 50%, after 24 hours of exposure. The three additives, in all treatments and times of analysis considered, caused significant inhibition of cell division in A. cepa, however did not cause cellular alterations to the evaluated meristems. These food flavorings also caused significant mortality to micro crustaceans with LC50<100 μg/mL. From this, under the conditions of mentioned analyzes, cherry, banana and hazelnut flavorings induced significant toxicity and cytotoxicity to the bioassays used.

En general, los aspectos toxicológicos de los saborizantes de los alimentos son poco estudiados. Esta condición justifica las evaluaciones de toxicidad, citotoxicidad y genotoxicidad de estas sustancias. En el presente estudio, se evaluó la toxicidad de los aromas de plátano, cereza y avellana en células meristemáticas de raíces de Allium cepa, en forma pura (según comercializa) y en concentraciones de 12.5; 25 y 50%, después de 24 y 48 horas de exposición. El potencial tóxico de estos aditivos alimentarios también se evaluó frente a nauplios de Artemia salina a concentraciones de 0,78; 1,56; 3.12; 6.25; 12.5; 25 y 50%, después de 24 horas de exposición. Los tres aditivos, en todos los tratamientos y tiempos de análisis considerados, causaron inhibición significativa de la división celular en A. cepa, sin embargo, no causaron alteraciones celulares a los meristemos evaluados. Estos saborizantes alimentarios también causaron una mortalidad significativa a microcrustáceos con LC50 <100 μg/ mL. A partir de esto, bajo las condiciones de los análisis descriptos, los aromatizantes de cereza, plátano y avellana indujeron toxicidad significativa y citotoxicidad para los bioensayos utilizados.

Aluminium-induced cell death requires upregulation of NtVPE1 gene coding vacuolar processing enzyme in tobacco (Nicotiana tabacum L.).

Cell death mechanism triggered by aluminium (Al) ion was investigated at root apex of tobacco (cultivar Bright Yellow) and in cultured tobacco cell line BY-2 derived from Bright Yellow, focusing on VPE genes (NtVPE1a, NtVPE1b, NtVPE2, NtVPE3). Cell death was detected as a loss of integrity of the plasma membrane by vital staining with fluorescein diacetate (in root apex) and Evans blue (in BY-2), respectively. At root apex, the upregulation of gene expression of VPE1a and VPE1b was observed significantly after 9h of Al exposure in parallel with an enhancement of cell death, while the upregulation of VPE2 and VPE3 were observed later. Similarly, in BY-2 cells, the upregulation of VPE1a and VPE1b and the enhancement of cell death were synchronously observed after 3-h exposure to Al, while the upregulation of VPE2 and VPE3 occurred later. RNA interference (RNAi) lines of each of the VPEs were constructed in BY-2 cells. Comparative studies between wild-type and the RNAi lines indicated that both Al-enhanced VPE activity and Al-induced cell death were significantly suppressed in the RNAi lines of VPE1 (dual suppressor of VPE1a and VPE1b), but not in the RNAi lines of VPE2 and that of VPE3. Taken together, we conclude that the upregulation of VPE1 gene expression and following enhancement of VPE activity under Al stress cause cell death in actively growing or elongating cells of tobacco.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.