Hydrogen peroxide modulates the expression of the target of rapamycin (TOR) and cell division in Arabidopsis thaliana.

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results.

Sucrose supplements modulate the Pseudomonas chlororaphis-Arabidopsis thaliana interaction via decreasing the production of phenazines and enhancing the root auxin response.

Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism, photosynthesis, biomass production and/or reinforcing immunity. However, information into how these microbes behave under different growth conditions is missing. In this work, we tested how carbon supplements modulate the interaction of Pseudomonas chlororaphis with Arabidopsis thaliana. P. chlororaphis streaks strongly repressed primary root growth, lateral root formation and ultimately, biomass production. Noteworthy, increasing sucrose availability into the media from 0 to 2.4% restored plant growth and promoted lateral root formation in bacterized seedlings. This effect could not be observed by supplementing sucrose to leaves only, indicating that the interaction was strongly modulated by bacterial access to sugar. Total phenazine content decreased in the bacteria grown in high (2.4%) sucrose medium, and conversely, the expression of phzH and pslA genes were diminished by sugar supply. Pyocyanin antagonized the promoting effects of sucrose in lateral root formation and biomass production in inoculated seedlings, indicating that this virulence factor accounts for growth repression during the plant-bacterial interaction. Defence reporter transgenes PR-1::GUS and LOX2::GUS were induced in leaves, while the expression of the auxin-inducible, synthetic reporter gene DR5::GUS was enhanced in the roots of bacterized seedlings at low and high sucrose treatments, which suggests that growth/defence trade-offs in plants are critically modulated by P. chlororaphis. Collectively, our data suggest that bacterial carbon nutrition controls the outcome of the relation with plants.

The HGF/Met Receptor Mediates Cytotoxic Effect of Bacterial Cyclodipeptides in Human Cervical Cancer Cells.

BACKGROUND: Human cervix adenocarcinoma (CC) caused by papillomavirus is the third most common cancer among female malignant tumors. Bioactive compounds such as cyclodipeptides (CDPs) possess cytotoxic effects in human cervical cancer HeLa cells mainly by blocking the PI3K/Akt/mTOR pathway and subsequently inducing gene expression by countless transcription regulators. However, the upstream elements of signaling pathways have not been well studied. METHODS: To elucidate the cytotoxic and antiproliferative responses of the HeLa cell line to CDPs by a transcriptomic analysis previously carried out, we identified by immunochemical analyses, differential expression of genes related to the hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/MET) receptors. Furthermore, molecular docking was carried out to evaluate the interactions of CDPs with the EGF and MET substrate binding sites. RESULTS: Immunochemical and molecular docking analyses suggest that the HGF/MET receptor participation in CDPs cytotoxic effect was independent of the protein expression levels. However, protein modulation of downstream Met-targets occurred due to the inhibition of phosphorylation of the HGF/MET receptor. Results suggest that the antiproliferative and cytotoxicity of CDPs in HeLa cells involve the HGF/MET receptor upstream of PI3K/Akt/mTOR pathway; assays with the human breast cancer MCF-7 and MDA-MB-231cell lines supported the finding. CONCLUSION: Data provide new insights into the molecular mechanisms involved in CDPs cytotoxicity and antiproliferative effects, suggesting that the signal transduction mechanism may be related to the inhibition of the phosphorylation of the EGF/MET receptor at the level of substrate binding site by an inhibition mechanism similar to that of Gefitinib and foretinib anti-neoplastic drugs.

Blood Serum Stimulates the Virulence Potential of Mucorales through Enhancement in Mitochondrial Oxidative Metabolism and Rhizoferrin Production.

This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.

Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Effect of Multi-walled Carbon Nanotubes Functionalized with Indol-3-butyric Acid on the Development of Avena sativa.

In pioneering research, it has been documented that the CNT influences the development of plants through the balance of phytoregulators. Therefore, in this work the objective is to evaluate the effects of CNT functionalized by non-covalent method with indole-3-butyric acid that they have on Avena sativa. The CNT was characterized by FTIR and Raman to confirm functionalization. It was observed that in the germination stage the seeds treated with IBA inhibited germination, however, when functionalizing the CNT with IBA it was observed that the CNT is contributing to counteract this inhibition.

Comparison of the Rhizobacteria Serratia sp. H6 and Enterobacter sp. L7 on Arabidopsis thaliana Growth Promotion.

The genera Serratia and Enterobacter belong to the Enterobacteriaceae family and several members have been described as plant growth-promoting rhizobacteria (PGPR). However, how these bacteria influence growth and development is unclear. We performed in vitro interaction assays between either Serratia sp. H6 or Enterobacter sp. L7 with Arabidopsis thaliana seedlings to analyze their effects on plant growth. In experiments of co-cultivation distant from the root tip, Enterobacter sp. decreased root length, markedly increased lateral root number, and slightly increased plant biomass by 33%, 230%, and 69%, respectively, and relative to the control. The volatile organic compounds (VOCs) emitted from Serratia sp. H6 but not those from Enterobacter sp. L7 promoted Arabidopsis growth. A blend of volatile compounds from the two bacteria had effects on plant growth that were similar to those observed for volatile compounds from H6 only. At several densities, the direct contact of roots with Serratia sp. H6 had phytostimulant properties but Enterobacter sp. L7 had clear deleterious effects. Together, these results suggest that direct contact and VOCs of Serratia sp. H6 were the main mechanisms to promote plant growth of A. thaliana, while diffusible compounds of Enterobacter sp. L7 were predominant in their PGPR activity.

Pathogenesis in Pseudomonas aeruginosa PAO1 Biofilm-Associated Is Dependent on the Pyoverdine and Pyocyanin Siderophores by Quorum Sensing Modulation.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals, and plants, through producing different molecular factors such as biofilm, siderophores, and other virulence factors which favor bacterial establishment and infection in the host. In P. aeruginosa PAO1, the production of these factors is regulated by the bacterial quorum sensing (QS) mechanisms. From them, siderophores are involved in iron acquisition, transport, and homeostasis. They are also considered some of the main virulence factors in P. aeruginosa; however, detailed mechanisms to induce bacterial pathogenesis are poorly understood. In this work, through reverse genetics, we evaluated the function of bacterial pathogenesis in the pvd cluster genes, which are required for synthesizing the siderophore pyoverdine (PVD). Single pvdI, pvdJ, pvdL, and double mutant strains were analyzed, and contrary to expected, the pvdL and pvdI mutations increased the concentration of PVD and other phenazines, such as pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) and also an increased biofilm production and morphology depending on the autoinducer 2-alkyl-4-quinolone (PQS) and the QS molecules acyl-homoserine lactones. Consequently, in the in vivo pathogenicity model of Caenorhabditis elegans, the mutations in pvdI, pvdJ, and pvdL increased the survival of the worms exposed to supernatants or biofilms of the bacterial cultures. However, the double mutant pvdI/pvdJ increased its toxicity in agreeing with the biofilm production, PVD, PYO, and PCA. The findings indicate that the mutations in pvd genes encode non-ribosomal peptide synthetases impacted the biofilm's structure, but suppressively also of the phenazines, confirming that the siderophores contribute to the bacterial establishment and pathogenicity of P. aeruginosa PAO1.

Linolenic Acid Plus Ethanol Exacerbates Cell Death in Saccharomyces cerevisiae by Promoting Lipid Peroxidation, Cardiolipin Loss, and Necrosis.

Polyunsaturated fatty acids (PUFA) hypersensitize yeast to oxidative stress. Ethanol accumulation during fermentation is another factor that induces oxidative stress via mitochondrial dysfunction and ROS overproduction. Since this microorganism has raised growing interest as a PUFA factory, we have studied if the combination of PUFA plus ethanol enhances yeast death. Respiration, ROS generation, lipid peroxidation, mitochondrial cardiolipin content, and cell death were assessed in yeast grown in the presence of 10% ethanol (ETOH) or linolenic acid (C18:3), or ethanol plus C18:3 (ETOH+C18:3). Lipid peroxidation and cardiolipin loss were several-fold higher in cells with ETOH+C18:3 than with C18:3. On the contrary, ETOH tended to increase cardiolipin content without inducing changes in lipid peroxidation. This was consistent with a remarkable diminution of cell growth and an exacerbated propidium iodide staining in cells with only ETOH+C18:3. The respiration rate decreased with all the treatments to a similar degree, and this was paralleled with similar increments in ROS between all the treatments. These results indicate that PUFA plus ethanol hypersensitize yeast to necrotic cell death by exacerbating membrane damage and mitochondrial cardiolipin loss, independent of mitochondrial dysfunction and ROS generation. The implications of these observations for some biotechnological applications in yeast and its physiology are discussed.

Bacterial cyclodipeptides elicit Arabidopsis thaliana immune responses reducing the pathogenic effects of Pseudomonas aeruginosa PAO1 strains on plant development.

Plants being sessile organisms are exposed to various biotic and abiotic factors, thus causing stress. The Pseudomonas aeruginosa bacterium is an opportunistic pathogen for animals, insects, and plants. Direct exposure of Arabidopsis thaliana to the P. aeruginosa PAO1 strain induces plant death by producing a wide variety of virulence factors, which are regulated mainly by quorum sensing systems. Besides virulence factors, P. aeruginosa PAO1 also produces cyclodipeptides (CDPs), which possess auxin-like activity and promote plant growth through activation of the target of the rapamycin (AtTOR) pathway. On the other hand, plant defense mechanisms are regulated through the production of phytohormones, such as salicylic acid (SA) and jasmonic acid (JA), which are induced in response to pathogen-associated molecular patterns (PAMPs), activating defense genes associated with SA and JA such as PATHOGENESIS-RELATED-1 (PR-1) and LIPOXYGENASE2 (LOX2), respectively. PR proteins are suggested to play critical roles in coordinating the Systemic Acquired Resistance (SAR). In contrast, LOX proteins (LOX2, LOX3, and LOX4) have been associated with the production of JA by producing its precursors, oxylipins. The activation of defense mechanisms involves signaling cascades such as Mitogen-Activated Protein Kinases (MAPKs) or the TOR pathway as a switch for re-directing energy towards defense or growth. In this work, we challenged A. thaliana (wild type, mpk6 or mpk3 mutants, and overexpressing TOR) seedlings with P. aeruginosa PAO1 strains to identify the role of bacterial CDPs in the plant immune response. Results showed that the pre-exposure of these Arabidopsis seedlings to CDPs significantly reduced plant infection of the pathogenic P. aeruginosa PAO1 strains, indicating that plants that over-express AtTOR or lack MPK3/MPK6 protein-kinases are more susceptible to the pathogenic effects. In addition, CDPs induced the GUS activity only in the LOX2::GUS plants, indicative of JA-signaling activation. Our findings indicate that the CDPs are molecules that trigger SA-independent and JA-dependent defense responses in A. thaliana; hence, bacterial CDPs may be considered elicitors of the Arabidopsis immune response to pathogens.