Impact of priming on seed germination, seedling growth and gene expression in common vetch under salinity stress.

Salinity is one of the most important abiotic stress factors that is expanding its influence because of global climate change and global warming. It causes gene expression changes, a reduction in seed germination and related characteristics, and poor seedling establishment in many crop plants by creating a lower osmotic potential in the seedbed and/or toxic ion effects in germinated seeds. In recent years, seed priming has been considered a promising strategy in modern stress management to protect plants against stress conditions. This study was conducted to elucidate the effects of osmopriming with polyethylene glycol 6000 (PEG-6000) on seed germination, seedling growth and gene expression in the common vetch (Vicia sativa L.) in different saline conditions. Common vetch seeds were primed with PEG-6000 solutions having different osmotic potentials (0.00, -0.50,  -0.75, -1.00, -1.25, and -1.50 MPa) for 12 hours. Control (un-primed) and primed seeds were germinated and seedlings were grown in different saline conditions (EC= zero, 4, 8 and 16 dS m-1). Furthermore, gene expression was compared in the primed seedlings in two different osmotic potentials (0.00 and -1.50 MPa) by microarray technology. Results demonstrated that germination percentage of common vetch seeds and seedling growth were diminished by high salinity. However, several priming treatments alleviated the adverse effects of high salinity on germination and early seedling growth of common vetch. The microarray showed that the expression of many genes in both stress and normal conditions was not significantly different.

Effect of herbicides in the oxidative stress in crop winter species.

Most herbicides applied in crop field, stay in the soil for a period, affecting next crop or even the plants using as green manure. Nowadays, the use of herbicides grow to increase productivity, mainly in the grain producing region north of Rio Grande do Sul state. The objective of this study was to evaluate the effects of herbicides fomesafen and sulfentrazone on antioxidant system in Avena sativa1, Vicia sativa2, Raphanus sativus and Lupinus albus. The plants were exposed to varying concentrations of fomesafen3 (0, 0.125, 0.25 and 0.5 kg ha -1) and sulfentrazone (0, 0.3, 0.6 and 1.2 kg ha-1). For this, the activities of, ascorbat peroxidase, catalase and guaiacol enzymes were analyzed, and the levels of MDA were quantificated. Fomesafen and sulfentrazone promoted alterations in balance of plants generating oxidative stress and elicited the response of the antioxidant system of plants, mainly in the high doses of fomesafen, for the species V. sativa and R. sativus. At the same time, the 1,2 kg ha -1 dose of sulfentrazone generated lipid peroxidation for V. sativa, R. sativus and L. albus. Additionally, A. sativa was the species that demonstrated low alterations on antioxidant system with the exposure to herbicide fomesafen and sulfentrazone. Thus, we can we can suggest that the species present a better response in defense of the oxidative stress generated by the herbicides.

Cd-induced oxidative stress and lignification in the roots of two Vicia sativa L. varieties with different Cd tolerances.

We examined the effects of Cd on growth, lipid peroxidation, reactive oxygen species (ROS) accumulation, antioxidant enzymatic activity, and lignin content in the roots of two varieties of Vicia sativa. Treatment with Cd decreased plant growth and increased ROS and lipid peroxidation levels to a greater extent in the Cd-sensitive variety ZM than in the Cd-tolerant variety L3. Most hydrogen peroxide (H2O2) and superoxide anion (O2(•-)) were accumulated in the cell walls and extracellular spaces in response to Cd treatments. Chemical assays and experiments using inhibitors showed that larger increases in H2O2 and O2(•-) production in ZM than in L3 were probably attributed to elevated Cd-induced nicotinamide adenine dinucleotide-peroxidase (NADH-POD) activity. Cd treatment increased the accumulation of lignin and the guaiacol peroxidase (GPOD) activities in the apoplast more significantly in ZM root than in L3. Howerver, root laccase activity was higher in L3 than in ZM. Thus Cd toxicity induced significant lignification in the roots of V. sativa, and increases in H2O2 accumulation and apoplastic GPOD activity were likely responsible for this effect.

Response to copper bromide exposure in Vicia sativa L. seeds: analysis of genotoxicity, nucleolar activity and mineral profile.

Copper bromide (CuBr2) effects on seed germination and plantlet development of Vicia sativa L. are evaluated through mitotic index, chromosome aberrations, nucleolar activity and mineral profile. CuBr2 induces a significant presence of micronuclei, sticky and c-metaphases, anaphase bridges and chromosome breaks. Increased number of nucleoli and scattering of AgNOR proteins from the nucleolus in the nuclear surface at CuBr2 1mM and in the cytoplasm at CuBr2 5mM, goes along with the decrease of root growth. In V. sativa embryo the content of many macro and micronutrients increases up to copper 1mM in agreement with reserve mobilization while at CuBr2 5mM some elements are present in lower amount. We hypothesize that inhibitory effects observed at 5mM are due either to a nutrient shortage or to a direct influence of copper on root cell division, evidenced by low mitotic index, high occurrence of chromosome aberrations and loss of material from the nucleolus.

Metabolic responses in root nodules of Phaseolus vulgaris and Vicia sativa exposed to the imazamox herbicide.

Alterations on growth, amino acids metabolism and some antioxidant enzyme activities as result of imazamox treatment were examined in determinate and indeterminate nodules, formed by Phaseolus vulgaris and Vicia sativa, respectively. Young seedlings of both legumes were inoculated with their respective microsymbionts and grown under controlled conditions. At vegetative growth, plants were treated with imazamox (250µM) in the nutrient solution and harvested 7days after. Imazamox was mainly accumulated in V. sativa where concentrations were more than six fold higher than those detected in P. vulgaris. Nodule dry weight and total nitrogen content were reduced by the herbicide treatment: the highest decrease of nodule biomass (50%) and nitrogen content (40%) were registered in V. sativa and P. vulgaris, respectively. The concentration of branched-chain amino acids (BCAA) did not change in neither determinate nor indeterminate nodules even though the acetohydroxyacid synthase activity decreased in root and nodules of both symbioses with the herbicide application. Based on this last result and taking into account that total free amino acids increased in roots but not in nodules of common vetch, a possible BCAA translocation from root to nodule could occur. Our results suggest that the maintenance of BCAA balance in nodule become a priority for the plant in such conditions. The involvement of activities glutathione-S-transferase, guaiacol peroxidase and superoxide dismutase in the response of the symbioses to imazamox are also discussed.

Physiological and biochemical responses of common vetch to the imazamox accumulation.

Common vetch (Vicia sativa L.) is a forage and grain legume, widely distributed throughout the world. Alterations induced by the herbicide imazamox on plant growth, acetohydroxyacid synthase activity, total free amino acids, as well as concentrations of valine, leucine, isoleucine and imazamox in young and mature leaves were investigated at 2 and 7 days after the herbicide application. Plant growth decreased significantly after 7 days of imazamox treatment. The herbicide was detected in both young and mature leaves inducing an inhibition of acetohydroxyacid synthase activity in the former and consequently decreasing valine and leucine contents in this organ. At the same time, the treatment caused an increase of total free amino acids in young leaves, presumably as result of proteolysis stimulation in such conditions. Given that these effects were not observed in mature leaves, we suggest a different sensitivity of the acetohydroxyacid synthase activity to imazamox depending on leaf age. Common vetch seems not to degrade imazamox since the herbicide was accumulated in shoot with increasing treatment time. To our knowledge, no physiological and biochemical studies of common vetch responses to imazamox have been previously reported.

Effects of copper on germination and reserve mobilization in Vicia sativa L. seeds.

The effects of different copper concentrations on percentage germination, increase in fresh weight and radicle growth of Vicia sativa L. seeds were studied. Physiological studies showed that the germination rate was not affected up to a concentration of 5 × 10⁻³ M, but already at 10⁻³ M the copper stopped root elongation. Structural and ultrastructural observations of embryo and cotyledon reserve mobilization showed that inhibition of radicle growth at 10⁻³ M Cu concentration cannot be ascribed to nutrient shortage but probably to an effect of copper on radicle cell division and elongation. In seeds treated with 5 × 10⁻³ M CuBr2, the copper completely inhibited cotyledon protein mobilization, so that embryo protein mobilization supported normal growth of the radicle up to 30 h after imbibition. The particular protein content of adjacent cotyledon cells is also discussed.

Plants as models for chromium and nickel risk assessment.

The adverse effects of Cr(III), Cr(VI), and Ni(II) expressed as root and shoot growth inhibition, metal accumulation and translocation throughout plants, and genotoxicity study were examined. To examine phytoxicity and metal accumulation, Vicia sativa, Raphanus sativus, Zea mays and Sinapis alba plants were used. Except for S. alba root growth inhibition, Ni had the strongest inhibitory effect on root and shoot growth. The inhibitory rank order based on IC50 values was Ni(II) > Cr(VI) > Cr(III). Z. mays was the least sensitive to all metals. While the accumulation of Cr was higher in the roots than the upper plant parts, Ni transport to shoots was at least two times higher than that of Cr. The highest accumulation of Cr was found in Z. mays and that of Ni in V. sativa and Z. mays roots. For all plants, the translocation factor was higher for Cr(VI) than for Cr(III). The translocation factor for Ni was several times higher than those of Cr. For mutagenicity assay, root tips of V. sativa, R. sativus and Z. mays were used. All metals exerted a significant increase of chromosomal aberrations and the rank order of aberrations was: Cr(VI) > Ni(II) > Cr(III). Genotoxic effects of metals were also determined by analysis of micronuclei frequency in the pollen tetrads of Tradescantia plants. None of metals significantly stimulated micronuclei frequency and the genotoxic effect decreased in the following order: Cr(VI) ≥ Ni(II) > Cr(III).

Phytoremediation of phenol using Vicia sativa L. plants and its antioxidative response.

Common vetch (Vicia sativa L.) is a legume species with an extensive agricultural use. However, the phytoremediation potentiality of this species has not been sufficiently explored because little is known about its resistance to inorganic and organic pollutants. In the present work, phenol tolerance of common vetch was assayed at different stages of growth. Germination index and germination rate decreased only at high phenol concentrations (250 and 500 mg L(-1)), whereas 30-day-old plants were able to tolerate this pollutant, with high removal efficiencies. The activities of antioxidative enzymes, such as peroxidase (POD) and ascorbate peroxidase, increased significantly with the highest phenol concentration, whereas superoxide dismutase activity, malondialdehyde, and H(2)O(2) levels remained unaltered. Besides, an increase in two basic isoforms of POD was observed in plants treated with phenol. The results suggested that common vetch has an efficient protection mechanism against phenol-induced oxidative damage. Moreover, it could tolerate and remove high phenol concentrations, avoiding serious phytotoxic effects. Thus, V. sativa could be considered an interesting tool in the field of phytoremediation.

Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplasts of Phaseolus aureus and Vicia sativa.

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 µM significantly increased the production of hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(·-) ) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H(2)O(2) and O(2)(·-) was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 µM SA decreased Cd-induced production of H(2)O(2) and O(2)(·-) in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.

Specific accumulation of CYP94A1 transcripts after exposure to gaseous benzaldehyde: induction of lauric acid ω-hydroxylase activity in Vicia sativa exposed to atmospheric pollutants.

The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96 ± 10 versus 12 ± 2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10-1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

Antigenotoxic effect of extract from Cynara cardunculus L.

The extract of artichoke Cynara cardunculus L. (CCE) was investigated for its potential antigenotoxic and antioxidant effects using four experimental model systems. In the Saccharomyces cerevisiae mutagenicity/antimutagenicity assay, CCE significantly reduced the frequency of 4-nitroquinoline-N-oxide-induced revertants at the ilv1 locus and mitotic gene convertants at the trp5 locus in the diploid Saccharomyces cerevisiae tester strain D7. In the simultaneous toxicity and clastogenicity/anticlastogenicity assay, it exerted an anticlastogenic effect against N-nitroso-N'-methylurea-induced clastogenicity in the plant species Vicia sativa L. On the contrary, despite CCE not being mutagenic itself, in the preincubation Ames assay with metabolic activation, it significantly increased the mutagenic effect of 2-aminofluorene in the bacterial strain Salmonella typhimurium TA98. In the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, CCE exhibited considerable antioxidant activity. The SC50 value representing 0.0054% CCE corresponds to an antioxidant activity of 216.8 microm ascorbic acid which was used as a reference compound. Although the mechanism of CCE action still remains to be elucidated, different possible mechanisms are probably involved in the CCE antigenotoxic effects. It could be concluded that CCE is of particular interest as a suitable candidate for an effective chemopreventive agent.

CYP94A1, a plant cytochrome P450-catalyzing fatty acid omega-hydroxylase, is selectively induced by chemical stress in Vicia sativa seedlings.

CYP94A1 is a cytochrome P450 (P450) catalyzing fatty acid (FA) omega-hydroxylation in Vicia sativa seedlings. To study the physiological role of this FA monooxygenase, we report here on its regulation at the transcriptional level (Northern blot). Transcripts of CYP94A1, as those of two other P450-dependent FA hydroxylases (CYP94A2 and CYP94A3) from V. sativa, are barely detectable during the early development of the seedlings. CYP94A1 transcripts, in contrast to those of the two other isoforms, are rapidly (less than 20 min) and strongly (more than 100 times) enhanced after treatment by clofibrate, an hypolipidemic drug in animals and an antiauxin (p-chlorophenoxyisobutyric acid) in plants, by auxins (2,4-dichlorophenoxyacetic acid and indole-3-acetic acid), by an inactive auxin analog (2,3-dichlorophenoxyacetic acid), and also by salicylic acid. All these compounds activate CYP94A1 transcription only at high concentrations (50-500 microM range). In parallel, these high levels of clofibrate and auxins modify seedling growth and development. Therefore, the expression of CYP94A1 under these conditions and the concomitant morphological and cytological modifications would suggest the implication of this P450 in a process of plant defense against chemical injury.

Antigenotoxic potential of glucomannan on four model test systems.

Antimutagenic, anticlastogenic, and bioprotective effect of polysaccharide glucomannan (GM) isolated from Candida utilis was evaluated in four model test systems. The antimutagenic effect of GM against 9-aminoacridine (9-AA)- and sodium azide (NaN3)-induced mutagenicity was revealed in the Salmonella typhimurium strains TA97 and TA100, respectively. GM showed anticlastogenic effect against N-nitroso-N'-methylurea (NMU) induced chromosome aberrations in the Vicia sativa assay. The bioprotective effect of GM co-treated with methyl-methane-sulphonate (MMS) was also established in Chlamydomonas reinhardtii repair deficient strains uvs10 and uvs14. The statistically significant antimutagenic potential of GM was not proved against 4-nitro-quinoline-1-oxide (4-NQO)-induced mutagenicity in Saccharomyces cerevisiae D7 assay. It may be due to bioprotectivity of alpha-mannan and beta-glucan, which are integral part of S. cerevisiae cell walls. Due to the good water solubility, low molecular weight (30 kDa), antimutagenic/anticlastogenic, and bioprotective activity against chemical compounds differing in mode of action, GM appears to be a promising natural protective (antimutagenic) agent.

The effect of diesel fuel on common vetch (Vicia sativa L.) plants.

When petroleum hydrocarbons contaminate soil, the carbon:nitrogen (C:N) ratio of the soil is altered. The added carbon stimulates microbial numbers but causes an imbalance in the C:N ratio which may result in immobilization of soil nitrogen by the microbial biomass, leaving none available for plant growth. As members of Leguminosae fix atmospheric nitrogen to produce their own nitrogen for growth, they may prove more successful at growing on petroleum hydrocarbon contaminated sites. During a wider study on phytoremediation of diesel fuel contaminated soil, particular attention was given to the performance of legumes versus other plant species. During harvesting of pot experiments containing leguminous plants, a recurring difference in the number and formation of root nodules present on control and contaminated Common vetch (Vicia sativa L.) plants was observed. The total number of nodules per plant was significant reduced in contaminated plants compared to control plants but nodules on contaminated plants were more developed than corresponding nodules on control plants. Plant performance of Common vetch and Westerwold's ryegrass (Lolium multiflorum L.) was compared to illustrate any difference between the ability of legumes and grasses to grow on diesel fuel contaminated soil. Common vetch was less affected by diesel fuel and performed better in low levels of diesel fuel contaminated soil than Westerwold's ryegrass. The total amount of diesel fuel remaining after 4 months in Common vetch planted soil was slightly less than in Westerwold's ryegrass planted soil.

Antimutagenic potential of homoisoflavonoids from Muscari racemosum.

The potential antimutagenic effect of the plant extract of Muscari racemosum bulbs, rich on 3-benzylidene-4-chromanones, was evaluated on three genetic model organisms. The mixture of three homoisoflavonoids was applied together with diagnostic mutagens in the Ames assay on four bacterial strains Salmonella typhimurium TA97, TA98, TA100, TA102, in the toxicity and mutagenicity/antimutagenicity assay on the yeast strain Saccharomyces cerevisiae D7, and in the simultaneous phytotoxicity and clastogenicity/anticlastogenicity assay on Vicia sativa (L.). The extract exerted antimutagenic and anticlastogenic effects due to the presence of homoisoflavonoids, which may be included in the group of natural antimutagens. This genotoxicological study suggests that homoisoflavonoids from M. racemosum (L.) owing to antimutagenic and anticlastogenic properties are of great pharmacological importance, and might be beneficial for prevention of cancer.