Eleazar Rodriguez

Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants.

Chromium (VI) induces toxicity at different photosynthetic levels in pea

In order to comprehensively characterize the effects of Cr (VI) on the photosynthetic performance of Pisum sativum, plants irrigated with Cr solutions (ranging from 20 to 2000 mg l(-1)) were evaluated using the following classical endpoints: gas exchange parameters, chlorophyll a (Chl a) fluorescence, leaf pigments, Rubisco activity, soluble sugars and starch content. Flow cytometry (FCM) was applied in an innovative approach to evaluate the morphological and fluorescence emission status of chloroplasts from plants exposed to Cr (VI). The parameters related to gas exchange, net CO(2) assimilation rate (A) and Rubisco activity were severally affected by Cr exposure, in some cases even at the lowest dosage used. While all biomarkers used to measure Chl a fluorescence indicated a decrease in fluorescence at the maximum dosage, pigment contents significantly increased in response to Cr (VI). The morphology of chloroplasts also was altered by Cr (VI) exposure, as a volume decrease was observed. Soluble sugars and starch showed an overall tendency to increase in Cr (VI) exposed plants, but sucrose and glucose decreased highly when exposed to 2000 mg l(-1). In conclusion, our results indicate that Cr (VI) affects photosynthesis at several levels, but the most Cr (VI)-sensitive endpoints were chloroplast morphology and biochemical processes; only at higher dosages the photochemical efficiency is compromised.

Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry

We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg).

Detection of somaclonal variants in somatic embryogenesis-regenerated plants of Vitis vinifera by flow cytometry and microsatellite markers

Flow cytometry and microsatellite analyses were used to evaluate the trueness-to-type of somatic embryogenesis-regenerated plants from six important Spanish grapevine (Vitis vinifera L.) cultivars. Tetraploid plants were regenerated through somatic embryogenesis from all of the cultivars tested with the exception of ‘Merenzao’. In addition, an octoploid plant was obtained in the cv. ‘Albariño’, and two mixoploids in ‘Torrontés’. The most probable origin of these ploidy variations is somaclonal variation. The cv. ‘Brancellao’ presented significantly more polyploids (28.57%) than any other cultivar, but it must be noted that 50% of the adult field-grown ‘Brancellao’ mother plants analysed were mixoploid. Hence, it is probable that these polyploids originated either from somaclonal variation or by separation of genotypically different cell layers through somatic embryogenesis. Microsatellite analysis of somatic embryogenesis-regenerated plants showed true-to-type varietal genotypes for all plants except six ‘Torrontés’ plants, which showed a mutant allele (231) instead of the normal one (237) at the locus VVMD5. There was not a clear relationship between the occurrence of the observed mutant regenerated plants and the callus induction media composition, the developmental stage of the inflorescences, the type of explant used for starting the cultures or the type of germination (precocious in differentiation medium or normal in germination medium) in any of the cultivars tested, except ‘Torrontés’. The mutant plants described herein have been transplanted to soil for future evaluation of putative phenotypic traits of interest. These mutants can be useful both for breeding programs and for functional genomic approaches aimed at increasing knowledge of the biology of grapevine.

Phytotoxicity of Mercury in Plants: A Review

With the advent of the industrial revolution, heavy metal contamination has become an ever increasing concern. Thus, it is of major importance to understand the extent of the toxicity in plants and animals and the consequences from the ingestion of contaminated food. Hg is easily modified into several oxidation states, and it can be spread in many ecosystems. Due to the recurrence of Hg pollution and due to the lack of knowledge about the effects of this heavy metal in plants, the aim of the present text is to provide a comprehensive review of the literature regarding Hg phytotoxicity.

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Review on Some Emerging Endpoints of Chromium (VI) and Lead Phytotoxicity

Metals occur naturally in the environment as constituents of the Earths crust and they tend to accumulate and persist due to their stability and mainly because they cannot be degraded or destroyed. However, and despite that in some cases (e.g. mercury) high levels occur naturally, for most situations, anthropic activities are among the primary causes for metal pollution. Examples of important sources of metal contamination come from industrial applications, mining, smelters, combustion by-products and fuel. From these sources, contaminants can enter the ecosystem as airborne particles, wastewaters and sludge, polluting not only sites near the source but locations thousands of kilometers apart. Studies like the ones of Murozumi et al. (1969), Hong et al. (1994) or McConnell and Edwards (2008) demonstrated the extension and persistence of metals in the environment. These studies also showed that contamination of the environment with these pollutants started way before the industrial revolution with evidence of pollution originating from Roman mining and smelters in 500 B.C. (Nriagu, 1996).

Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxification capacities.

Cadmium (Cd) is a widespread environmental contaminant, strongly mutagenic and known to cause DNA damage in plants. In this work, flow cytometry (FCM) was applied to determine if in vivo exposure to Cd would induce genotoxic effects at the genome level. The hyper-accumulator Thlaspi caerulescens (J. & C. Presl), the related non-accumulator Thlaspi arvense L. and the accumulator crop species Lactuca sativa L. were germinated in distilled water and grown in modified Hoaglands medium with increasing concentrations of Cd(NO3)2 (0, 1, 10 and 100 microM). After 28 days of exposure, shoot and root growth was recorded and the tissues were harvested for Cd and FCM analysis. In general, roots from treated plants contained higher content of Cd than leaves and growth inhibition was observed in the treated plants. Nuclear DNA content was estimated and the G0/G1 full peak coefficient of variation (FPCV), as an indicator of clastogenic damage, was recorded. In T. arvense and T. caerulescens no significant differences were detected between control and exposed plants. Leaves of L. sativa exposed to 10 microM Cd presented a statistically significant increase in FPCV values in comparison with the control group. Furthermore, roots exposed to 100 microM Cd presented a reduction in nuclear DNA content and an increase in FPCV when compared to the control. FCM data indicates that no major DNA damage was induced on both Cd-exposed Thlaspi species and L. sativa leaves. On the contrary, results obtained with L. sativa roots suggests clastogenic damage in these organs exposed to 100 microM of Cd.

Transcript profiling and DNA damage in the European eel (Anguilla anguilla L.) exposed to 7,12-dimethylbenz[a]anthracene

The molecular responses induced during and after an acute exposure to 7,12-dimethylbenz[a]anthracene (DMBA) were analysed in liver, gill and blood cells of juvenile Anguilla anguilla with the aim of developing molecular biomarkers of environmental PAH pollution. Changes in the mRNA expression levels of the cell cycle checkpoint-related rad1 gene and the mRNAs of differentially expressed genes by suppression subtractive hybridization (SSH) were analysed in the liver, and related to well-established biomarkers: cyp1A1 mRNA expression and assessment of the DNA integrity using the comet assay and flow cytometry. DMBA exposure resulted in increased cyp1A1 mRNA levels, suggesting that cyp1A1 might be involved in the metabolism of DMBA. Global DNA damage, detected by the comet assay, was observed in the three tissues analysed but only blood cells showed chromosomal lesions as analysed by flow cytometry. Although DNA damage was found in the liver, no induction in rad1 gene was observed in this organ. The global SSH approach revealed that mRNAs of genes related to xenobiotic metabolism, immune processes and cytoskeleton dynamics were differentially expressed in DMBA-exposed eel livers, highlighting the complexity in the response observed in fish exposed to a genotoxic agent and providing directions for new biomarker development.

Chloroplast functionality assessment by flow cytometry: Case study with pea plants under Paraquat stress

Photosynthesis is one of the most important processes in plant biology and in the development of new methodologies that allow a better understanding and characterization of the photosynthetic status of organisms, which is invaluable. Flow cytometry (FCM) is an excellent tool for measuring fluorescence and physical proprieties of particles but it has seldom been used in photosynthetic studies and thus the full extent of its potentialities, in this field of research, remains unknown. To determine the suitability of FCM in photosynthesis studies, pea plants were exposed to Paraquat and their status was analyzed during 24 h. FCM was used to evaluate the integrity (volume and internal complexity) and the relative fluorescence intensity (FL) of chloroplasts extracted from those plants. To elucidate which type of information the FL conveys, FL values were correlated with the minimum fluorescence level (F0), maximum fluorescence level (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), obtained by using Pulse-Amplitude-Modulation (PAM) fluorometry. Results indicate that: (1) the biomarkers used to evaluate the structural integrity of the chloroplasts were more sensitive to Paraquat exposure than the ones related to fluorescence; (2) the variation of the chloroplast’s structure, as time progressed, pointed to a swelling and subsequent burst of the chloroplast which, in turn, compromised fluorescence emission; (3) FL presented a high and significant correlation with the Fv/Fm and to a lesser degree with Fm but not with F0; (4) pigment content did not reveal significant changes in response to Paraquat exposure and is in agreement with the proposed model, suggesting that the cause for fluorescence decrease is due to chloroplast disruption. In sum, FCM proved to be an outstanding technique to evaluate chloroplastidal functional and structural status and therefore it should be regarded as a valuable asset in the field of photosynthetic research.