Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips

Cytotoxicity of metals is important because some metals are potential mutagens able to induce tumors in humans and experimental animals. Chromium can damage DNA in several ways, including DNA double strand breaks (DSBs) which generate chromosomal aberrations, micronucleus formation, sister chromatid exchange, formation of DNA adducts and alterations in DNA replication and transcription. In our study, water samples from three sites in the Córrego dos Bagres stream in the Franca municipality of the Brazilian state of São Paulo were subjected to the comet assay and micronucleus test using erythrocytes from the fish Oreochromis niloticus. Nuclear abnormalities of the erythrocytes included blebbed, notched and lobed nuclei, probably due to genotoxic chromium compounds. The greatest comet assay damage occurred with water from a chromium-containing tannery effluent discharge site, supporting the hypothesis that chromium residues can be genotoxic. The mutagenicity of the water samples was assessed using the onion root-tip cell assay, the most frequent chromosomal abnormalities observed being: c-metaphases, stick chromosome, chromosome breaks and losses, bridged anaphases, multipolar anaphases, and micronucleated and binucleated cells. Onion root-tip cell mutagenicity was highest for water samples containing the highest levels of chromium.

Investigation on the s of guava (Psidium guajava L.) infusions on germination, root tips and meristematic cells of Latuca sativa

Guava (Psidium guajava L.) is a plant often employed in popular medicine. Recently several studies have alerted about the toxicity of substances present in medicinal plants, which can pose risks to the human health. In this sense, the present work aimed to investigate the phytotoxic, cytotoxic and genotoxic action of three guava varieties - Paluma, Pedro Sato and Roxa (purple) - on the plant test system Lactuca sativa L. Thus, macro- and microscopic evaluations were carried out for five infusion concentrations (2.5, 5.0, 10.0, 20.0 and 40.0 g.L-1) prepared from each variety. Distilled water was used as negative control. Chromatographic and spectroscopic analysis by HPLC-PAD indicated that the chemical composition of the infusion of Roxa is different than that of the infusions of the varieties Paluma and Pedro Sato. It was observed that seed germination and root growth in L. sativa exposed to infusions decreased with increasing infusion concentration, regardless of the tested cultivar. For the mitotic index, no statistical differences were observed. On the other hand, a significant increase in the frequency of cell cycle alterations was verified, especially for the highest concentrations tested. The cytogenotoxic was significant. Therefore, guava should not be used indiscriminately in popular medicine.

High Aluminum Resistance in Buckwheat1 : I. Al-induced Specific Secretion of Oxalic Acid from Root Tips

High Al resistance in buckwheat (Fagopyrum esculentum Moench. cv Jianxi) has been suggested to be associated with both internal and external detoxification mechanisms. In this study the characteristics of the external detoxification mechanism, Al-induced secretion of oxalic acid, were investigated. Eleven days of P depletion failed to induce secretion of oxalic acid. Exposure to 50 μm LaCl3 also did not induce the secretion of oxalic acid, suggesting that this secretion is a specific response to Al stress. Secretion of oxalic acid was maintained for 8 h by a 3-h pulse treatment with 150 μm Al. A nondestructive method was developed to determine the site of the secretion along the root. Oxalic acid was found to be secreted in the region 0 to 10 mm from the root tip. Experiments using excised roots also showed that secretion was located on the root tip. Four kinds of anion-channel inhibitors showed different effects on Al-induced secretion of oxalic acid: 10 μm anthracene-9-carboxylic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate had no effect, niflumic acid stimulated the secretion 4-fold, and phenylglyoxal inhibited the secretion by 50%. Root elongation in buckwheat was not inhibited by 25 μm Al or 10 μm phenylglyoxal alone but was inhibited by 40% in the presence of Al and phenylglyoxal, confirming that secretion of oxalic acid is associated with Al resistance.

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

Adventitious root formation in anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as 'difficult'- or 'easy-to-root' when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be 'difficult' precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting. In this study we used softwood cuttings of cashew (Anacardium occidentale), a species considered as 'difficult-to-root'. We aimed to test the hypothesis that speed, and extent of early rooting, is critical in determining success with this species; and that the potential to form adventitious roots will decrease with time in the propagation environment. Using two genotypes, initial rooting rates were examined in the presence or absence of exogenous auxin. In cuttings that formed adventitious roots, either entire roots or root tips were removed, to determine if further root formation/development was feasible. To investigate if subsequent root responses were linked to phytohormone action, a number of cuttings were also treated with either exogenous auxin (indole-3-butyric acid-IBA) or cytokinin (zeatin). Despite the reputation of Anacardium as being 'difficult-to-root', we found high rooting rates in two genotypes (AC 10 and CCP 1001). Removing adventitious roots from cuttings and returning them to the propagation environment, resulted in subsequent re-rooting. Indeed, individual cuttings could develop new adventitious roots on four to five separate occasions over a 9 week period. Data showed that rooting potential increased, not decreased with time in the propagation environment and that cutting viability was unaffected. Root expression was faster (8-15 days) after the removal of previous roots compared to when the cuttings were first stuck (21 days). Exposing cuttings to IBA at the time of preparation, improved initial rooting in AC 10, but not in CCP 1001. Application of IBA once roots had formed had little effect on subsequent development, but zeatin reduced root length and promoted root number and dry matter accumulation. These results challenge our hypothesis, and indicate that rooting potential remains high in Anacardium. The precise mechanisms that regulate the number of adventitious roots expressed, remain to be determined. Nevertheless, results indicate that rooting potential can be high in 'difficult-to-root' species, and suggest that providing supportive environments is the key to expressing this potential. (c) 2006 Elsevier B.V. All rights reserved.

The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene

Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups. © 2013 Elsevier B.V.

Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems

Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, 63Ni and 65Zn were selectively redistributed within the plant. From autoradiographs, it became evident that 65Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while 63Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes).

Genetic ablation of root cap cells in Arabidopsis

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

Quantitative Intercellular Localization of NADH-Dependent Glutamate Synthase Protein in Different Types of Root Cells in Rice Plants1

The quantitative analysis with immunogold-electron microscopy using a single-affinity-purified anti-NADH-glutamate synthase (GOGAT) immunoglobulin G (IgG) as the primary antibody showed that the NADH-GOGAT protein was present in various forms of plastids in the cells of the epidermis and exodermis, in the cortex parenchyma, and in the vascular parenchyma of root tips (<10 mm) of rice (Oryza sativa) seedlings supplied with 1 mm NH4+ for 24 h. The values of the mean immunolabeling density of plastids were almost equal among these different cell types in the roots. However, the number of plastids per individual cell type was not identical, and some parts of the cells in the epidermis and exodermis contained large numbers of plastids that were heavily immunolabeled. Although there was an indication of labeling in the mitochondria using the single-affinity-purified anti-NADH-GOGAT IgG, this was not confirmed when a twice-affinity-purified IgG was used, indicating an exclusively plastidial location of the NADH-GOGAT protein in rice roots. These results, together with previous work from our laboratory (K. Ishiyama, T. Hayakawa, and T. Yamaya [1998] Planta 204: 288–294), suggest that the assimilation of exogeneously supplied NH4+ ions is primarily via the cytosolic glutamine synthetase/plastidial NADH-GOGAT cycle in specific regions of the epidermis and exodermis in rice roots. We also discuss the role of the NADH-GOGAT protein in vascular parenchyma cells.

Flavonoids Promote Haustoria Formation in the Root Parasite Triphysaria versicolor1

Parasitic plants in the Scrophulariaceae develop infective root structures called haustoria in response to chemical signals released from host-plant roots. This study used a simple in vitro assay to characterize natural and synthetic molecules that induce haustoria in the facultative parasite Triphysaria versicolor. Several phenolic acids, flavonoids, and the quinone 2,6-dimethoxy-p-benzoquinone induced haustoria in T. versicolor root tips within hours after treatment. The concentration at which different molecules were active varied widely, the most active being 2,6-dimethoxy-p-benzoquinone and the anthocyanidin peonidin. Maize (Zea mays) seeds are rich sources of molecules that induce T. versicolor haustoria in vitro, and chromatographic analyses indicated that the active molecules present in maize-seed rinses include anthocyanins, other flavonoids, and simple phenolics. The presence of different classes of inducing molecules in seed rinses was substantiated by the observation that maize kernels deficient in chalcone synthase, a key enzyme in flavonoid biosynthesis, released haustoria-inducing molecules, although at reduced levels compared with wild-type kernels. We discuss these results in light of existing models for host perception in the related parasitic plant Striga.

Meristem-Specific Suppression of Mitosis and a Global Switch in Gene Expression in the Root Cap of Pea by Endogenous Signals1

Two functionally distinct sets of meristematic cells exist within root tips of pea (Pisum sativum): the root apical meristem, which gives rise to the body of the root; and the root cap meristem, which gives rise to cells that differentiate progressively through the cap and separate ultimately from its periphery as border cells. When a specific number of border cells has accumulated on the root cap periphery, mitosis within the root cap meristem, but not the apical meristem, is suppressed. When border cells are removed by immersion of the root tip in water, a transient induction of mitosis in the root cap meristem can be detected starting within 5 min. A corresponding switch in gene expression throughout the root cap occurs in parallel with the increase in mitosis, and new border cells begin to separate from the root cap periphery within 1 h. The induction of renewed border cell production is inhibited by incubating root tips in extracellular material released from border cells. The results are consistent with the hypothesis that operation of the root cap meristem and consequent turnover of the root cap is self-regulated by a signal from border cells.

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root1

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.

Expressed Sequence Tags from a Root-Hair-Enriched Medicago truncatula cDNA Library1

The root hair is a specialized cell type involved in water and nutrient uptake in plants. In legumes the root hair is also the primary site of recognition and infection by symbiotic nitrogen-fixing Rhizobium bacteria. We have studied the root hairs of Medicago truncatula, which is emerging as an increasingly important model legume for studies of symbiotic nodulation. However, only 27 genes from M. truncatula were represented in GenBank/EMBL as of October, 1997. We report here the construction of a root-hair-enriched cDNA library and single-pass sequencing of randomly selected clones. Expressed sequence tags (899 total, 603 of which have homology to known genes) were generated and made available on the Internet. We believe that the database and the associated DNA materials will provide a useful resource to the community of scientists studying the biology of roots, root tips, root hairs, and nodulation.

Identification of auxin-responsive elements of parB and their expression in apices of shoot and root.

Detailed analysis of transgenic tobaccos containing a series of chimeric parB promoter/beta-glucuronidase (GUS) gene constructs allowed us to define two auxin-responsive elements (AREs) of 48 bp and 95 bp (positions -210 to -163 and -374 to -280) in the parB promoter. The two AREs responded independently to physiological concentrations of auxin. Gel retardation assays revealed binding of nuclear protein(s) to the sequence conserved between ARE I and ARE II. The auxin responsiveness of the parB promoter did not mediate the pathway through the as-1 element and transcription factor ASF-1. AREs I and II were responsive to auxin at physiological concentrations, whereas as-1 responded only to higher concentrations of auxin which may be interpreted as stress, though as-1 had been reported to be a minimal ARE [Liu, X. & Lam, E. (1994) J. Biol. Chem. 269, 668-675]. Histochemical staining of transgenic tobacco that contained a parB promoter/GUS construct demonstrated the expression of GUS activity in the shoot apex as well as in the root tips, suggesting the involvement of parB expression in meristematic activity or differentiation. The drastic change in auxin responsiveness in the transgenic plants between the 6th and 10th day after imbibition of seeds implies the development or the activation of auxin signal transduction systems during plant development.