Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Environmental effects on photosynthetic capacity of bean genotypes

Photosynthetic responses to daily environmental changes were studied in bean (Phaseolus vulgaris L.) genotypes 'Carioca', 'Ouro Negro', and Guarumbé. Light response curves of CO2 assimilation and stomatal conductance (g s) were also evaluated under controlled (optimum) environmental condition. Under this condition, CO2 assimilation of 'Carioca' was not saturated at 2,000 µmol m-2 s-1, whereas Guarumbé and 'Ouro Negro' exhibited different levels of light saturation. All genotypes showed dynamic photoinhibition and reversible increase in the minimum chlorophyll fluorescence yield under natural condition, as well as lower photosynthetic capacity when compared with optimum environmental condition. Since differences in g s were not observed between natural and controlled conditions for Guarumbé and 'Ouro Negro', the lower photosynthetic capacity of these genotypes under natural condition seems to be caused by high temperature effects on biochemical reactions, as suggested by increased alternative electron sinks. The highest g s values of 'Carioca' were observed at controlled condition, providing evidences that reduction of photosynthetic capacity at natural condition was due to low g s in addition to the high temperature effects on the photosynthetic apparatus. 'Carioca' exhibited the highest photosynthetic rates under optimum environmental condition, and was more affected by daily changes of air temperature and leaf-to-air vapor pressure difference.

Photosynthetic responses of young cashew plants to varying environmental conditions

The aim of this study was to characterize gas exchange responses of young cashew plants to varying photosynthetic photon flux density (PPFD), temperature, vapor-pressure deficit (VPD), and intercellular CO2 concentration (Ci), under controlled conditions. Daily courses of gas exchange and chlorophyll a fluorescence parameters were measured under natural conditions. Maximum CO2 assimilation rates, under optimal controlled conditions, were about 13 mmol m-2 s-1 , with light saturation around 1,000 mmol m-2 s-1. Leaf temperatures between 25ºC and 35ºC were optimal for photosynthesis. Stomata showed sensitivity to CO2, and a closing response with increasing Ci. Increasing VPD had a small effect on CO2 assimilation rates, with a small decrease above 2.5 kPa. Stomata, however, were strongly affected by VPD, exhibiting gradual closure above 1.5 kPa. The reduced stomatal conductances at high VPD were efficient in restricting water losses by transpiration, demonstrating the species adaptability to dry environments. Under natural irradiance, CO2 assimilation rates were saturated in early morning, following thereafter the PPFD changes. Transient Fv/Fm decreases were registered around 11h, indicating the occurrence of photoinhibition. Decreases of excitation capture efficiency, decreases of effective quantum yield of photosystem II, and increases in non-photochemical quenching were consistent with the occurrence of photoprotection under excessive irradiance levels.

Low temperature impact on photosynthetic parameters of coffee genotypes

The objective of this work was to evaluate photoprotective mechanisms related to low positive temperatures in Coffea canephora (Conilon clones 02 and 153) and C. arabica ('Catucaí' IPR 102) genotypes, involved in cold temperature tolerance. To accomplish this, one-year-old plants were successively submitted to: temperature decrease of 0.5ºC day-1, from 25/20ºC to 13/8ºC; a three-day chilling cycle at 13/4ºC; and a recovery period of 14 days (25/20ºC). During the experiment, leaf gas exchange, chlorophyll a fluorescence and leaf photosynthetic pigment content were evaluated. Total activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and ribulose-5-phosphate kinase (Ru5PK) were quantified to measure the activity of photosynthesis key enzymes. All genotypes showed low temperature sensitivity, but displayed diverse cold impact and recovery capabilities regarding the photosynthetic-related parameters studied. Catucaí IPR 102 cultivar showed better ability to cope with cold stress than the Conilon clones, especially Conilon 02, and had full recovery of leaf gas exchange, fluorescence parameters, enzymatic activity, and higher contents of the photoprotective pigments zeaxanthin and lutein.

Anatomy, chlorophyll content and photosynthetic potential in grapevine leaves under plastic cover

The present study evaluated the anatomy, chlorophyll content and photosynthetic potential of grapevine leaves grown under plastic cover. The experiment was carried out in vineyards of Moscato Giallo cultivar covered and uncovered with plastic. A block design with 10 selected plants was used for each area (covered and uncovered). Twelve leaves (six of them fully exposed to solar radiation and six grown under shaded conditions) were collected from each area and were fixed and analyzed microscopically (thickness of the adaxial and abaxial epidermis and of the palisade and spongy parenchymas). Chlorophyll content and photosynthetic potential were determined in the vineyard at veraison and after harvest. Plastic covering increased the thickness of the palisade parenchyma in exposed and shaded leaves due to solar radiation restriction. However, the leaves from the covered vineyard did not have the same response to the restriction of solar radiation, as observed in the uncovered vineyard. The thickness of the adaxial and abaxial epidermis and of the spongy parenchyma did not vary due to solar radiation restriction. Chlorophyll content increased in the leaves of covered plants. The photosynthetic potential of the vines is not affected by solar radiation restriction imposed by plastic cover due to anatomical modification in leaves.

Resposta antioxidante, formação de fitoquelatinas e composição de pigmentos fotoprotetores em Brachiaria decumbens Stapf submetida à contaminação com Cd e Zn

In order to evaluate the response of Brachiaria decumbens Stapf to Cd and Zn, plants were hydroponically exposed to 50 and 100 mmol L-1 of Cd and 500 and 2000 mmol L-1 of Zn. Metal content of shoots and roots was determined, as well as alterations in photosynthetic and photoprotective pigments, antioxidant metabolites and phytochelatin synthesis. Plants concentrated elevated levels of Cd and Zn, especially in roots. Zinc exposure negatively affected chlorophyll and β-carotene content, whereas the highest dose of Cd reduced VAZ cycle pigments and tocopherol levels in plant shoots. Cadmium was the maximum inducer of the phytochelatin synthesis pathway.

Sugarcane yellow leaf virus infection leads to alterations in photosynthetic efficiency and carbohydrate accumulation in sugarcane leaves

Infection by Sugarcane yellow leaf virus (ScYLV) causes severe leaf symptoms in sugarcane (Saccharum spp.) hybrids, which indicate alterations in its photosynthetic apparatus. To gain an overview of the physiological status of infected plants, we evaluated chlorophyll a fluorescence and gas exchange assays, correlating the results with leaf metabolic surveys, i.e., photosynthetic pigments and carbohydrate contents. When compared to healthy plants, infected plants showed a reduction in potential quantum efficiency for photochemistry of photosystem (PSII) and alterations in the filling up of the plastoquinone (PQ) pool. They also showed reduction in the CO2 net exchange rates, probably as a consequence of impaired quantum yield. In addition, reductions were found in the contents of photosynthetic leaf pigments and in the ratio chlorophyll a/chlorophyll b (chla/chlb). Carbohydrate content in the leaves was increased as a secondary effect of the ScYLV infection. This article discusses the relation of virus replication and host defense responses with general alterations in the photosynthetic apparatus and in the metabolism of infected plants.

Photosynthetic performance of mangroves Rhizophora mangle and Laguncularia racemosa under field conditions

In mature mangrove plants Rhizophora mangle L. and Laguncularia racemosa Gaerth. growing under field conditions, photosystem 2 (PS2) photochemical efficiency, determined by the ratio of variable to maximum fluorescence (Fν/Fm), increased during the day in response to salinity in the rainy seasons. During the dry season, fluorescence values (Fo) were higher than those observed in rainy season. In addition, Fo decreased during the day in both season and species, except for R. mangle during the dry season. A positive correlation among Fν/Fm and salinity values was obtained for R. mangle and L. Racemosa during the dry and rainy seasons, showing that photosynthetic performance is maintained in both species under high salinities. Carotenoid content was higher in L. Racemosa in both seasons, which represents an additional mechanism against damage to the photosynthetic machinery. The chlorophyll content was not affected by salinity in either species.

Metabolism and gene polymorphisms of the folate pathway in Brazilian women with history of recurrent abortion

PURPOSE: To investigate the association between polymorphisms in genes that encode enzymes involved in folate- and vitamin B12-dependent homocysteine metabolism and recurrent spontaneous abortion (RSA).METHODS: We investigated the C677T and A1298C polymorphisms of the methylenetetrahydrofalate reductase gene (MTHFR), the A2756G polymorphism of the methionine synthase gene (MS) and the 844ins68 insertion of the cystathionine beta synthetase gene (CBS). The PCR technique followed by RFLP was used to assess the polymorphisms; the serum levels of homocysteine, vitamin B12 and folate were investigated by chemiluminescence. The EPI Info Software version 6.04 was used for statistical analysis. Parametric variables were compared by Student's t-test and nonparametric variables by the Wilcoxon rank sum test.RESULTS: The frequencies of gene polymorphisms in 89 women with a history of idiopathic recurrent miscarriage and 150 controls were 19.1 and 19.6% for the C677T, insertion, 20.8 and 26% for the A1298C insertion, 14.2 and 21.9% for the A2756G insertion, and 16.4 and 18% for the 844ins68 insertion, respectively. There were no significant differences between case and control groups in any of the gene polymorphisms investigated. However, the frequency of the 844ins68 insertion in the CBS gene was higher among women with a history of loss during the third trimester of pregnancy (p=0.003). Serum homocysteine, vitamin B12 and folate levels id not differ between the polymorphisms studied in the case and control groups. However, linear regression analysis showed a dependence of serum folate levels on the maintenance of tHcy levels.CONCLUSION: The investigated gene polymorphisms and serum homocysteine, vitamin B12 and folate levels were not associated with idiopathic recurrent miscarriage in the present study. Further investigations are needed in order to confirm the role of the CBS 844ins68 insertion in recurrent miscarriage.

Photosynthetic activity of cassava plants under weed competition

The objective of this work was to evaluate characteristics associated with the photosynthetic activity of cassava plants under weed competition. The trial was carried out under field conditions, and experimental units consisted of 150 dm³ fiberglass boxes containing red yellow Latosol, previously corrected and fertilized. Treatments consisted in the cultivation of cassava plants which were free of weed competition and associated with three weed species: Bidens pilosa, Commelina benghalensis or Brachiaria plantaginea. After manioc sprouting started, 15 days after being planted, weeds that had been sown when manioc was planted were thinned, there were then eight plants left per experimental unit in accordance with specified treatments: cassava free of competition, cassava competing with B. pilosa, cassava competing with C. benghalensis and cassava competing with B. plantaginea. Sixty days after crop emergence leaf internal CO2 concentration (Ci), leaf temperature at the time of evaluation (Tleaf) and photosynthetic rate (A) were evaluated, also the CO2 consumption rate (ΔC) of cassava plants was calculated. A correlation matrix between variables was also obtained. All characteristics associated with photosynthesis in cassava plants were influenced by weed species. Cassava was more affected by B. pilosa and B. plantaginea in which concerns its exposition to solar radiation and water, while C. benghalensis seems to mostly affect the composition of incident light on the culture, allowing cassava to anticipate imposition when competing, even before it reaches harmful levels.

Photosynthetic characteristics of hybrid and conventional rice plants as a function of plant competition

The objective of this work was to evaluate the characteristics related to the photosynthetic ability of hybrid and inbred rice varieties, as a way to assess which of the two presented higher potential to stand out under conditions of competition. The trial was set in a greenhouse in completely randomized block design and 2 x 6 factorial scheme with four replications. Factor A consisted of rice varieties (hybrid or inbred) and factor B by competition levels. Treatments consisted in maintaining one plant of either BRS Pelota (inbred) or Inov (hybrid) variety at the center of the plot, under competition with 0, 1, 2, 3, 4 or 5 plants of the variety BRS Pelota at the periphery of the experimental unit, according to the treatment. Fifty days after emergence (DAE), sub-stomatal CO2 concentration (Ci - mmol mol-1), photosynthetic rate (A - mmol m-2 s-1) and CO2 consumed (DC - mmol mol-1) were quantified, as well as shoot dry mass(SDM).Hybrid plants present higher photosynthesis capacity than inbred plants, when competing with up to 3 times its own density. When under the same competitive intensity, hybrid plants surpass the inbred. However, it should be emphasized that, when in farm condition, the lower competitive capacity with weeds often attributed to the hybrid varieties, probably is due to their lower planting density, but if weed competition is kept at low levels, hybrid rice plants may perform in the same way or usually better than inbred plants.

Determination of photosynthetic pigments in fleabane biotypes susceptible and resistant to the Herbicide Glyphosate

Chlorophylls and carotenoids are the main photosynthetic pigment in plants. In the weeds, the greatest amount of photosynthetic pigments can result in high competitiveness of the species. The aim of this study was to quantify the content of photosynthetic pigments in biotypes of fleabane (Conyza bonariensis) susceptible and resistant to glyphosate, by two different methods, as well as a correlation between chlorophyll content obtained by portable and classical methodology (extractable chlorophyll). An experiment was conducted in greenhouse and laboratory, 2 x 5 factorial scheme, where factor A was equivalent to biotypes of fleabane (resistant and susceptible to glyphosate) and factor B to developmental stages plants (rosette vegetative I, II and III and reproduction). At all stages of development, fleabane plants were evaluated with the portable determiner (chlorophyll content) and then the same leaves were subjected to classical methodology laboratory (extractable pigments). The resistant biotype of fleabane showed higher contents of chlorophyll a, b, and total carotenoids, inferring a greater competitive potential regarding the susceptible population to the herbicide. The portable determiner of chlorophyll showed high correlation with the classical method of determination of photosynthetic pigments, and can thus be used to accurately assess this, saving time and reagents.

Extraction and Simultaneous Determination of Glyphosate, AMPA and Compounds of the Shikimic Acid Pathway in Plants

This study has aimed to develop a method for simultaneous extraction and determination by liquid chromatography and mass spectrometry (LC-MS/MS) of glyphosate, aminomethylphosphonic acid (AMPA), shikimic acid, quinic acid, phenylalanine, tyrosine and tryptophan. For the joint analysis of these compounds the best conditions of ionization in mass spectrometry and for chromatographic separation of the compounds were selected. Calibration curves and linearity ranges were also determined for each compound. Different extraction systems of the compounds were tested from plant tissues collected from sugarcane (Saccharum officinarum) and eucalyptus (Eucalyptus urophylla platiphylla) plants two days after the glyphosate application at the dose of 720 g a.e. ha-1. The plant material was dried in a forced air circulation drying oven and in a lyophilizer, and subsequently the extractions with acidified water (pH 2.5), acetonitrile-water (50:50) [v/v] and methanol-water (50:50) [v/v] were tested. To verify the recovery of the compounds in the plant matrix with acidified water as an extracting solution, the samples were fortified with a solution containing the mixture of the different analytical standards present so that this one presented the same levels of 50 and 100 μg L-1 of each compound. All experiments were conducted with three replicates. The analytical method developed was efficient for compounds quantifications. The extraction from the samples dried in an oven and using acidified water allowed better extraction levels for all compounds. The recovery levels of the compounds in the fortified samples with known amounts of each compound for both plants samples were rather satisfactory.