Conceição Santos

Assessment of biomarkers of cadmium stress in lettuce.

Laboratory and field studies have provided encouraging insights into the capacity of plants to act as biomonitors of environmental quality through the use of biomarkers. However, a better understanding of the overall process of Cd-induced senescence, describing the cascade of Cd effects in plants is needed for a selection of relevant biomarkers of Cd stress. In order to approach this, 5-week old Lactuca sativa L. were exposed for 14 days to 100muM Cd(NO(3))(2) and harvested at days 0, 1, 3, 7 and 14. The parameters measured included classical endpoints (shoot and root growth) and biochemical endpoints related to photosynthesis, nutrients content, and oxidative stress. Cadmium-exposed plants displayed nutrient imbalances in leaves and roots. Photosynthetic efficiency was significantly decreased and lipid peroxidation was enhanced. Antioxidant enzymes were significantly altered during exposure-catalase was inhibited by the end of exposure and peroxidase was induced at day 1 in young leaves. These alterations culminated in a decrease in shoot growth after 14-days exposure to Cd. Biochemical alterations could be used in integrative approaches with classical endpoints in ecotoxicological tests for Cd and after further testing in real scenarios conditions, they could form the basis of a plant biomarkers battery for monitoring and predicting early effects of exposure to Cd.

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.

Adverse effects of cadmium exposure on mouse sperm

The effects of cadmium chloride exposure on sperm functional parameters were evaluated on eight-week-old ICR-CD1 male mice administered with a single s.c. injection of 1, 2 and 3 mg CdCl(2)/kg bw. Groups of animals treated with each dose, as well as their respective controls, were sacrificed after 24h to detect short-term (acute) effects and after 35 days. Sperm cells were collected from the epididymis and several parameters of sperm quality and function were evaluated, namely density, morphology, motility, viability, mitochondrial function, acrosome integrity, together with DNA fragmentation assessed by the TUNEL assay. The short-term effects of cadmium chloride resulted in an increased fraction of sperm with abnormal morphology, premature acrosome reaction and reduced motility. Late term effects (after 35 days) included a drastic reduction of sperm cell numbers and sperm motility. An increase in DNA fragmentation was also detected.

Factors influencing somatic embryogenesis induction in Eucalyptus globulus Labill.: basal medium and anti-browning agents

The low induction rates of somatic embryogenesis (SE) in Eucalyptusglobulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.

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).

Flow cytometric and cytogenetic analyses of Iberian Peninsula Festuca spp.

Festuca L. has an important diversification centre in the Iberian Peninsula. We used chromosome counting, fluorescence (FISH) and genomic in situ hybridization (GISH), and DNA flow cytometry (FCM) to clarify the taxonomic position of several taxa, to search for phylogenetic relationships and to assess the extent and pattern of genome variation in fescues. The chromosome number of Festuca duriotagana var. barbata is determined for the first time and new ploidy level estimations are given for F. rothmaleri and F. summilusitana. In the latter species, besides the reported decaploid level, dodecaploidy was found in some populations, which points to the existence of an unrecognized taxon. Moreover, these differences were confirmed by FCM and a high positive correlation was found with the type of substrate where F. summilusitana was growing. For each section, a decrease of genome size with increase of polyploidy was observed. In general, in situ hybridization techniques failed to reveal phylogenetic relationships among the selected species. In FISH, a variation in the number of rDNA sites was observed in some species. GISH results indicate that F. henriquesii is not a progenitor of the studied polyploid species.

Nutrient responses and glutamate and proline metabolism in sunflower plants and calli under Na2SO4 stress

The growth of Helianthus annuus L. calli and plants was reduced in the presence of Na2SO4 (10, 25, 50, and 100 mM). SO42— and Na concentrations increased in stressed calli and plants while NO3—, Cl—, P, K, and Mg decreased in stressed plants and Ca in shoots. Stressed calli showed decreases of NO3—, Ca, K, and Mg concentrations. Calli adapted to 50 mM Na2SO4 accumulated more K and Ca and less ammonium than stressed non-adapted calli. Proline exhibited increases in stressed calli and plants that were accompanied by decreases of proline oxidase activities while pyrroline-5-carboxylate reductase (P5CR) and ornithine aminotransferase (OAT) activities increased. Adapted calli accumulated more proline and had higher P5CR and OAT activities than stressed non-adapted calli. Glutamate concentration decreased with stress, together with a stimulation of cytosolic glutamine synthetase (GS1) and a decrease of plastidal GS (GS2) activity. These data strongly suggest that the increase of P5CR and GS1 activities are responsible for the decrease of glutamate concentration leading, together with the stimulation of OAT and the inhibition of the proline oxidation metabolism, to an increase of proline levels in Na2SO4-stressed sunflower cells. These data also show that salt stress increases the release of endogenous ammonium and suggests that the increase of GS1 activity plays an important role in its elimination. Nahrstoffgehalte und Glutamat- und Prolinstoffwechsel in Sonnenblumenpflanzen und Calli unter Na2SO4-Stress Das Wachstum von Helianthus annuus L. Pflanzen und deren Calli wird in Gegenwart steigender Na2SO4-Belastung (10, 25, 50 bzw. 100 mM) gehemmt. Sowohl in gestressten Pflanzen wie in den Calli waren die SO4- und Na-Konzentrationen erhoht. In den gestressten Pflanzen waren die Konzentrationen von NO3., Cl, P, K und Mg in Wurzel und Spross, die von Ca lediglich im Spross gesenkt, wahrend in gestressten Calli NO3, Ca, K und Mg erniedrigt waren. Calli, die uber 8 Monate an 50 mM Na2SO4 adaptiert waren, akkumulierten unter Salzstress im Vergleich zu nicht-adaptierten mehr K und Ca, aber weniger Ammonium. Am starksten stieg in den Pflanzen sowie Calli unter Salzstress die Prolin-Konzentration, bedingt durch einen Abfall der Prolinoxidase-Aktivitat sowie einen Anstieg der Pyrrolin-5-carboxylat-Reductase (P5CR) und der Ornithin-Aminotransferase (OAT). Die an Salinitat adaptierten Calli akkumulierten mehr Prolin und zeigten hohere Aktivitaten an P5CR und OAT. Bei Salzstress nahm die Konzentration an Glutamat ab. Dies war begleitet von einer Stimulierung der cytosolischen Glutaminsynthetase (GS1), jedoch einem Abfall der plastidaren GS2. Auf Grund der Ergebnisse kann der Anstieg der Prolingehalte nach Salzstress durch die Anstiege in der Aktivitat von P5CR und GS1, die die Absenkung der Glutamatkonzentration bewirken, sowie von OAT und durch die Hemmung der Prolinoxidase erklart werden. Der durch Salzstress ebenfalls geforderten Freisetzung endogenen Ammoniums wirkt die gesteigerte GS1-Aktivitat entgegen.

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.

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

SummarySomatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl−1 sucrose, 3 gl−1 gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.

Is the interplay between epigenetic markers related to the acclimation of cork oak plants to high temperatures

Trees necessarily experience changes in temperature, requiring efficient short-term strategies that become crucial in environmental change adaptability. DNA methylation and histone posttranslational modifications have been shown to play a key role in both epigenetic control and plant functional status under stress by controlling the functional state of chromatin and gene expression. Cork oak (Quercus suber L.) is a key stone of the Mediterranean region, growing at temperatures of 45°C. This species was subjected to a cumulative temperature increase from 25°C to 55°C under laboratory conditions in order to test the hypothesis that epigenetic code is related to heat stress tolerance. Electrolyte leakage increased after 35°C, but all plants survived to 55°C. DNA methylation and acetylated histone H3 (AcH3) levels were monitored by HPCE (high performance capillary electrophoresis), MS-RAPD (methylation-sensitive random-amplified polymorphic DNA) and Protein Gel Blot analysis and the spatial distribution of the modifications was assessed using a confocal microscope. DNA methylation analysed by HPCE revealed an increase at 55°C, while MS-RAPD results pointed to dynamic methylation-demethylation patterns over stress. Protein Gel Blot showed the abundance index of AcH3 decreasing from 25°C to 45°C. The immunohistochemical detection of 5-mC (5-methyl-2′-deoxycytidine) and AcH3 came upon the previous results. These results indicate that epigenetic mechanisms such as DNA methylation and histone H3 acetylation have opposite and particular dynamics that can be crucial for the stepwise establishment of this species into such high stress (55°C), allowing its acclimation and survival. This is the first report that assesses epigenetic regulation in order to investigate heat tolerance in forest trees.