Allelochemical Stress Can Trigger Oxidative Damage in Receptor Plants: Mode of Action of Phytotoxicity

Plants can interact with other plants through the release of chemical compounds or allelochemicals. These compounds released by donor plants influence germination, growth, development, and establishment of receptor plants; having an important role on the pattern of vegetation, i.e as invasive strategy, and on crop productivity. This phytotoxic or negative effect of the released allelochemicals (allelochemical stress) is caused by modifying or altering diverse metabolic processes, having many molecular targets in the receptor plants. Recently, using an aggressive and allelopathic plant Sicyos deppei as the donor plant, and Lycopersicon esculentum as the receptor plant, we showed that the allelochemicals released by S. deppei caused oxidative damage through an increase in reactive oxygen species (ROS) and activation or modification of antioxidant enzymes. Based on this study, we proposed that oxidative stress is one of the mechanisms, among others, by which an allelopathic plant causes phytotoxicity to other plants.Key Words: allelochemical stress, Sicyos deppei, Lycopersicon esculentum, plant allelochemicals, phytotoxicity, ROS, lipid peroxidationIt is well known that plants interact with many organisms, including co-habitation with other plants. Among these relations are the ones referred to as allelochemical interactions. Allelopathy can be defined as a mechanism of interference in plant growth and development mediated by the addition of plant-produced secondary products (allelochemicals) to the soil rhizosphere. Allelochemicals are present in all types of plants and tissues and are released into the soil rhizosphere by a variety of mechanisms, including decomposition of residues, volatilization, and root exudation.^1–3 These released allelochemicals become stressful only when they are toxic or when they affect the growth and development of surrounding plants (phytotoxicity). Studies on allelochemical stress have been expanding; recently the phenomenon has taken on increased importance, since it can help explain plant growth inhibition in interspecies interactions and in structuring the plant community. It appears to be one mechanism or strategy used by invasive plants to become successful and replace other native ones.^4–6On the other hand, the chemical diversity of the organic compounds that mediate these allelochemical interactions is as diverse as their modes of action. Many studies have shown that allelochemicals interfere with several physiological processes in the receptor organism.^3,7,8 The physiological effects on receptor plants or other organisms are useful in determining the role of the allelochemicals in the system. Recently, it has been proposed that allelochemicals can cause oxidative stress in target plants and therefore activate the antioxidant mechanism.^3,8–12 In particular; our studies have been focused on knowing the physiological targets of the phytotoxic compounds released by a noxious and endemic weed Sicyos deppei G. Don (Cucurbitaceae). We have taken as the model the receptor or damaged plant Lycopersicon esculentum Mill (Solanaceae), since in Mexican crop-fields, it is common to find both plants. We have observed the strong allelopathic potential of S. deppei and are exploring the potential metabolic target that could be involved in the strong phytotoxic effect of this weed.^13–16 We recently documented the oxidative damage that an aqueous leachate of S. deppei caused in the target plant L. esculentum.¹⁶ In this work we explored in seeds and in primary roots the antioxidant mechanism of tomato to determine whether or not the inhibitory effect of S. deppei was due to oxidative damage. We analyzed the activity and expression of some antioxidant enzymes involved in the detoxification of ROS, and found an imbalance in its activity as well as an increase in the levels of H₂O₂ at 24 h of treatment. Additional studies on the levels of ROS, including hydrogen peroxide, were monitored in primary roots from germinating seeds under allelochemical stress by imaging the ROS-sensitive fluorescent dye dichlorofluorescein (_H2DCFDA, carboxy-2′, 7′-diclhlorofluorescein diacetate) in a confocal microscope (BIORAD 1024, 488 nm dichroic and 510–560 nm emission). DCFDA fluorescence increases as the dye is oxidized by ROS to dichlorofluorescein (DCF). Figure 1 shows a marked increase in fluorescence at 48 h and 72 h of treatment (Fig. 1A–C) compared with the same treatment at 24 h, and with the corresponding control. This fluorescence was more evident at the root cap and at the zone of root hairs in treated seeds.Open in a separate windowFigure 1Allelochemical stress caused by S. deppei elicits ROS generation in tomato germinating seeds. Panels show control (left) and treatment (right) at 24 h (A), 48 h (B), and 72 h (C). Lower panels show higher magnification (40X) of the corresponding time. Seedlings with primary roots were stained for 10–15 minutes with 25 µM DCFDA in distilled water.Clearly, allelochemical stress caused by S. deppei is producing an oxidative imbalance as evidenced by generation of ROS and alteration of activity of antioxidant enzymes. Another result that supports this observation is the high level of lipid peroxidation that we observed at 48 and 72 h, which correlates with the inhibition of two membrane-associated enzymes, H⁺-ATPase¹⁵ and NADPH oxidase.¹⁶ We believe, however, that the oxidative damage we observed is not solely responsible for the phytotoxic effect of S. deppei on tomato growth. In other words, we suggest that its inhibitory effect represents the sum of many metabolic processes affected at different times. Currently we are studying the dynamics of carbohydrate mobilization, cell wall loosing of the endosperm to allow the protrusion of the radicle, and ABA content. Preliminary results have shown that there is a delay in expression of some enzyme activities and a high content of ABA.     

Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to alpha-factor

The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The alpha-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFalpha1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATalpha, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Galpha-GTPase activity, showed increased sensitivity to synthetic alpha-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in alpha-pheromone proteolysis) showed delay in mating but sensitivity to alpha-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaealpha-pheromone, whereas the MATalpha cell produces the a-pheromone.     

Alterations in ultrastructural morphology of two-cell bovine embryos produced in vitro and in vivo following a physiologically relevant heat shock

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at approximately 28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5 degrees C (cow body temperature), 41.0 degrees C (characteristic temperature for heat-stressed cows), or 43.0 degrees C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0 degrees C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5 degrees C or 41.0 degrees C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.     

Immunological adjuvants efficiently induce antigen-specific T cell responses in old mice: implications for vaccine adjuvant development in aged individuals

The morbidity and mortality of infectious diseases are significantly increased in aged humans. Hence, vaccination has been suggested as a means to reduce or prevent the impact of infections on old individuals. However, it has remained unresolved whether or not standard vaccine adjuvants such as aluminum hydroxide (Alum) are similarly efficacious in old individuals, as compared to young adults. Here, we have investigated the effects of prototypic immunological adjuvants, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), or Alum on HEL-specific T cell responses in young adult and old mice. We report that independent of the adjuvant used, the induced T cell responses to the prototypic protein antigen hen eggwhite lysozyme (HEL) were similar in young adult and old mice in terms of cytokine production, T cell frequencies, determinant specificity, and T cell repertoire. The results suggest that vaccine adjuvants developed in young adults should be equally effective in inducing T cell immunity in old individuals.     

Sequential Statistical Improvement of the Liquid Cultivation of Helicobacter pylori

Background: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. Materials and Methods: Four statistical models were sequentially used. First, a Box‐Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D‐optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box‐Behnken design was used to optimize the concentration of the culture media components previously selected. Results: After 12 hours of liquid culture a concentration of 25 × 10⁸ cells per mL (9.4 log₁₀ cells per mL) of H. pylori was obtained, compared with a predicted 32 × 10⁸ (9.5 log₁₀ cells per mL), which means between 1 and 5 log₁₀ units higher than some previous reports. Conclusions: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb’s blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 °C with filtered CO₂ (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.     

G6PD Deficiency and Hemoglobinopathies: Molecular Epidemiological Characteristics and Healthy Effects on Malaria Endemic Bioko Island,Equatorial Guinea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies were the inherited conditions found mostly in African. However, few epidemiological data of these disorders was reported in Equatorial Guinea (EQG). This study aimed to assess the prevalence and healthy effects of G6PD deficiency and hemoglobinopathies among the people on malaria endemic Bioko Island, EQG.

Materials and Methods

Blood samples from 4,144 unrelated subjects were analyzed for G6PD deficieny by fluorescence spot test (FST), high-resolution melting assay and PCR-DNA sequencing. In addition, 1,186 samples were randomly selected from the 4,144 subjects for detection of hemoglobin S (HbS), HbC, and α-thalassemia deletion by complete blood count, PCR-DNA sequencing and reverse dot blot (RDB).

Results

The prevalence of malaria and anemia was 12.6% (522/4,144) and 32.8% (389/1,186), respectively. Overall, 8.7% subjects (359/4,144) were G6PD-deficient by FST, including 9.0% (249/2,758) males and 7.9% (110/1,386) females. Among the 359 G6PD-deficient individuals molecularly studied, the G6PD A^- (G202A/A376G) were detected in 356 cases (99.2%), G6PD Betica (T968C/A376G) in 3 cases. Among the 1,186 subjects, 201 cases were HbS heterozygotes, 35 cases were HbC heterozygotes, and 2 cases were HbCS double heterozygotes; 452 cases showed heterozygous α-thalassemia 3.7 kb deletion (-α^3.7 kb deletion) and 85 homozygous - α^3.7 kb deletion. The overall allele frequencies were HbS 17.1% (203/1186); HbC, 3.1% (37/1186); and –α^3.7 kb deletion 52.4% (622/1186), respectively.

Conclusions

High G6PD deficiency in this population indicate that diagnosis and management of G6PD deficiency is necessary on Bioko Island. Obligatory newborn screening, prenatal screening and counseling for these genetic disorders, especially HbS, are needed on the island.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.