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Main conclusion: Higher vacuolar proton pump activity may increase plant energy and nutrient use efficiency and provide the nexus between plant inoculation with Herbaspirillum seropedicae and growth promotion. Abstract: Global change and growing human population are exhausting arable land and resources, including water and fertilizers. We present inoculation with the endophytic plant-growth promoting bacterium (PGPB) Herbaspirillum seropedicae as a strategy for promoting growth, nutrient uptake and photosynthetic efficiency in rice (Oryza sativa L.). Because plant nutrient acquisition is coordinated with photosynthesis and the plant carbon status, we hypothesize that inoculation with H. seropedicae will stimulate proton (H+) pumps, increasing plant growth nutrient uptake and photosynthetic efficiency at low nutrient levels. Plants were inoculated and grown in pots with sterile soil for 90 days. Herbaspirillum seropedicae endophytic colonization was successful and, as hypothesized, inoculation (1) stimulated root vacuolar H+ pumps (vacuolar H+-ATPase and vacuolar H+-PPase), and (2) increased plant growth, nutrient contents and photosynthetic efficiency. The results showed that inoculation with the endophytic bacterium H. seropedicae can promote plant growth, nutrient uptake and photosynthetic efficiency, which will likely result in a more efficient use of resources (nutrients and water) and higher production of nutrient-rich food at reduced economic and environmental costs.

The objective of the present study was to evaluate the antimicrobial activity of the Cc-LTP2 and Cc-GRP peptides isolated from Coffea canephora seeds and their possible synergistic activity with the azole drug fluconazole and characterize their mechanisms of action on cells of pathogenic fungi. Cc-LTP2 and Cc-GRP alone or in combination with 20 µg/mL of fluconazole were evaluated for their antimicrobial activity on the fungus Fusarium solani, and the effects of these peptides on the permeability of membranes and the induction of oxidative stress were determined. Our results show that these peptides at a concentration of 400 µg/mL combined with 20 µg/mL of fluconazole were able to inhibit the growth of the tested fungi, promote changes in their growth pattern, permeabilize the membrane, and induce reactive oxygen species (ROS). Some of these results were also observed with the peptides alone or with fluconazole alone, suggesting that the peptides act synergistically, promoting the potentiation of antimicrobial action. In this study, it was shown that Cc-LTP2 and Cc-GRP in combination with fluconazole were able to inhibit the growth of the fungus F. solani, to promote permeabilization of its membrane, and to induce the production of ROS, suggesting a combinatorial activity between the peptides and fluconazole.

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 μM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

CaThi is a thionin-like peptide isolated from fruits of Capsicum annuum, which has strong antimicrobial activity against bacteria, yeasts and filamentous fungi, and induced reactive oxygen species (ROS) in fungi. ROS are molecules that appear in the early stages of programmed cell death or apoptosis in fungi. Due to this fact, in this work we analyzed some events that may be related to process of apoptosis on yeast induced by CaThi. To investigate this possibility, we evaluated phosphatidylserine (PS) externalization, presence of active caspases and the ability of CaThi to bind to DNA in Candida tropicalis cells. Additionally, we investigated mitochondrial membrane potential, cell surface pH, and extracellular H+ fluxes in C. tropicalis cells after treatment with CaThi. Our results showed that CaThi induced PS externalization in the outer leaflet of the cell membrane, activation of caspases, and it had the ability for DNA binding and to dissipate mitochondrial membrane potential. In addition, the cell surface pH increased significantly when the C. tropicalis cells were exposed to CaThi which corroborates with ~96% inhibition on extracellular H+ efflux. Taking together, these data suggest that this peptide is capable of promoting an imbalance in pH homeostasis during yeast cell death playing a modulatory role in the H+ transport systems. In conclusion, our results strongly indicated that CaThi triggers apoptosis in C. tropicalis cells, involving a pH signaling mechanism.

Background Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice. Methods The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach. Results The inhibition of V-ATPases by 100 μM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4–5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers. Conclusions These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition. General significance The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential.

We tested the legacy of the farming system (conventional or organic) on the interactions among phosphate solubilizing bacteria (PSB) and phosphate (Pi) solubilisation. We determined the in vitro Pi solubilisation and production of organic acids by nine PSB (four from conventional and five from organic farming) isolated from the rhizospheres of Carica papya, grown in monoculture or in pairs (66 combinations). When grown in monoculture, PSB isolated from conventional farming solubilised more Pi, but when grown in pairs, these PSB solubilised the least Pi. Furthermore, when the pairs included only PSB isolated from conventional farming, no Pi-solubilising cooperation was observed, that is, Pi solubilisation by pairs was lower than in monoculture. When PSB isolated from organic farming were present in the pairs, ∼40% of the combinations resulted in Pi solubilising cooperation. Organic acid production was not related with Pi solubilisation. Our work demonstrated the existence of a farming system legacy influencing the biotic interactions among PSB, and that the output of the interactions (cooperation or antagonism) was not phylogenetically determined. Data highlighted that biotic interactions should be considered when screening for potential plant growth promoting bacteria.

Background V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane. Methods Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). Results Cholesterol depletion by 5 mM MβCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MβCD. Conclusions We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. General significance The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma.

Crop management systems influence plant productivity and nutrient use efficiency, as well as plant growth-promoting rhizobacteria (PGPR), which are known to influence the growth of plants via phytohormone production, phosphate solubilization, nitrogen (N) fixation and antimicrobial activity. The objective of this study was to compare the influence of two crop management system on microbial PGPR features. PGPR isolated from the rhizospheres of Carica papaya L. grown under two distinct management systems (conventional and organic) were identified and characterized. The 12 strains most efficient in solubilizing inorganic phosphate belonged to the genera Burkholderia, Klebsiella, and Leclercia. N fixation was observed in the strains B. vietnamiensis from the conventional farming system and B. vietnamiensis, B. cepacia and Leclercia sp. from the organic farming system. The B. vietnamiensis, B. cepacia, Klebsiella sp. and Klebsiella sp. isolates showed antifungal activity, while Leclercia sp. did not. The strains B. vietnamiensis and Enterobcter sp. (isolated from the conventional farming system) and Klebsiella sp. (isolated from the organic farming system) were efficient at solubilizing phosphate, producing phytohormones and siderophores, and inhibiting the mycelial growth of various phytopathogenic fungi (Botrytis cinerea, Pestalotia sp., Alternaria sp., Phoma sp., Fusarium culmorum, Geotrichum candidum). Physiological differences between the isolates from the two crop management regimes were distinguishable after 10 years of distinct management.