Herbaspirillum seropedicae is a plant growth-promoting bacterium that senses root exudates, colonizes the rhizosphere, attaches to the root surface and establishes endophytically in cereals. However, how these events reshape host physiological responses of maize roots and early microbiome assembly remains unclear. We investigated the metabolic and physiological responses of maize roots to inoculation with H. seropedicae HRC54 and assessed the consequences for the seed-resident/root-associated bacteriome. Gene expression of tricarboxylic acid (TCA) cycle enzymes was quantified by RT-qPCR. Rhizospheric H+ fluxes and surface pH were mapped using the non-invasive scanning ion-selective electrode technique (SIET). Root-zone attachment was visualized using scanning electron microscopy (SEM), and the bacterial community composition was profiled through 16S rRNA sequencing. Inoculation upregulated transcripts for aconitase, citrate synthase, isocitrate dehydrogenase, and succinate-CoA ligase, while downregulating fumarase and malate dehydrogenase. H+ efflux increased in the elongation zone and decreased in the root cap and root-hair zones, corresponding to localized pH shifts. SEM revealed preferential colonization of the elongation zone, matching localized pH shifts. SEM showed preferential colonization of the elongation zone, progressing from aggregates to biofilm within 24 h. Microbiome analysis revealed distinct beta-diversity and enrichment of genera such as Aurantimonas, Mesorhizobium, Novosphingobium, Serratia, and Stenotrophomonas, as well as a reduced abundance of several genera, including Bradyrhizobium, Burkholderia, and Gluconacetobacter. These results link TCA reprogramming to pH modulation and early microbiome reshaping, supporting seed-treatment strategies that enhance nutrient uptake, bolster resilience against root pathogens, and improve crop performance.

This study simultaneously evaluated fermentation kinetics and H+ fluxes during fermentation by Saccharomyces cerevisiae in order to elucidate the biochemical mechanisms behind bioethanol production induced by ELF magnetic fields. Fermentation kinetics were monitored by glucose uptake, ethanol production, secondary products (glycerol), pH, biomass, and biochemical characterization of ATPase activity through proton efflux density measurements. A face-centered central composite design 22 was performed, considering independent variables such as recycling arrangements (bioreactor with a spiral-shaped tube, whole bioreactor, and bioreactor with a U-shaped tube) and magnetic flux density (5, 10, and 15 mT), while alcoholic fermentation efficiency and H+ efflux density of the P-ATPase were the response variables. ELF magnetic fields induced substrate consumption and bioethanol formation in all experimental configurations when compared to fermentations without the field. However, the whole bioreactor exposed to 10 mT was the most attractive condition, resulting in an average increase of approximately 40 % and 18 % in the overall volumetric productivity of glucose consumption and ethanol production, respectively, compared to the control experiment. The effect of magnetic flux density on fermentative efficiency and H+ efflux density was statistically significant (p < 0.1). A direct correlation between H+ efflux and yeast performance during fermentation under magnetic fields was found through the application of a method for simultaneous optimization of the experimental results. The data are discussed in relation to the biological mechanism of action involved and the potential applications of this technology at an industrial scale are envisioned.

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD – Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-β-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations.

Depletion of the stratospheric ozone layer enhances UV exposure to living organisms. We hypothesized that the adaptative physiological and bioenergetic responses of modern genotypes of Coffea arabica cv. Catuaí Amarelo IAC 62, and C. canephora (Conilon LB1) to actual UV levels would generate additional costs to the detriment of biomass formation and partitioning. Coffee plants were cultivated for six months in a mini greenhouse under either near ambient (UVam) or reduced (UVre) UV conditions, after which leaf gas exchanges, chlorophyll a fluorescence parameters and proton pump phosphohydrolase activities were analyzed. Growth under UVam did not affect the photochemical efficiency and net CO2 assimilation in both Coffea species, but reduced stomatal conductance (gs) and increased intrinsic water use efficiency (iWUE), maximum photosynthetic O2 evolution, and apparent quantum yield. Coffee plants under UVam showed decreased P-type H+-ATPase activity, whilst H+-PPase activity increased, preserving metabolic energy. Coffee biomass accumulation decreases under UVam were more pronounced in C. canephora, which generally invested more in the root system than in shoot biomass as compared to C. arabica. Reduced biomass in C. canephora under UVam susuggested restrained C-sequestration in plant biomass, which might be partly associated with gs reduction, along with a equilibrium between the ATP- and PPi-driven electrochemical H+ gradients, providing a mechanism for energy buffering and cellular robustness. The stability of fluorescence parameters and net CO2 assimilation rate under UVam, reduced gs and increased iWUE, oxygen-evolving complex activity, and elevated SPAD values, supported photomorphogenetic responses acclimation to actual UV in two modern coffee genotypes, with detrimental impact on shoot biomass, especially in C. canephora.

Comparative analysis of expression patterns of ATP6V1C1 encoding C1 subunit of V-H+-ATPase revealed that molecular alterations correlated with endometrial cancer of better prognosis were grouped with low ATP6V1C1 expression while those correlated with worse prognosis were clustered with high ATP6V1C1 expression levels. Expression patterns of C1 subunit in endometrial adenocarcinoma are associated with molecular malignancy signatures shared by histological subtypes of highest mortality rate suggesting that G3 adenocarcinoma exhibits molecular changes resembling endometrial serous carcinoma. ATP6V1C1 might serve as novel prognostic marker allowing identification of targetable pathways for high-risk endometrial cancer.

The aim of this work was to evaluate the bioethanol productivity in an unconventional bioreactor assisted by extremely low frequency (ELF) - electromagnetic field and elucidate the biophysical mechanism of action by which ELF magnetic fields improve the bioethanol production by S. cerevisiae. Fermentations were carried out under axial field lines at 10 mT magnetic flux density (B), using three different recycling arrangements (spiral-shape tube, u-shape tube and whole bioreactor) in a closed loop. Fermentation kinetics were monitored by cell growth, substrate consumption, ethanol and by-product formation. In addition, electrophysiological measurements of the H+ ion fluxes were carried out in yeast cells sampled at different fermentation stages. ELF magnetic fields increased the glucose uptake, bioethanol production and H+ efflux, shortening in 2 h the fermentation time. The greatest effects of the ELF magnetic fields were obtained in the whole bioreactor arrangement, reaching an average increase of 33% in the bioethanol production. The results are consistent with a stimulatory effect of ELF magnetic fields on the plasma membrane H+-ATPase activity, as indicated by the specific increase of the vanadate-sensitive component of the yeast cells H+ efflux, providing a new biophysical mechanism of action for the biological effect of magnetic fields. (C) 2021 Elsevier Ltd. All rights reserved.

Estima-se que por ano cerca de 34.800 dos casos de câncer sejam associados a infeccao persistente por HPV de alto risco. Um dos principais mecanismos pelo qual o HPV induz transformacao celular e atraves das oncoproteinas virais, como a E6 que degrada a proteina supressora tumoral, p53. No nucleo, essa proteina esta envolvida principalmente na parada do ciclo celular e morte celular programada. No citoplasma, onde sua atuacao e menos conhecida, p53 interage com a Ca2+-ATPase de reticulo endoplasmatico (SERCA). Em celulas normais, sob estresse, p53 interage com a SERCA, promovendo ativacao desta bomba e aumentando a translocacao de Ca2+ do citoplasma para o reticulo endoplasmatico. O aumento de Ca2+ no reticulo induz o fluxo desse ion do reticulo para a mitocondria, culminando em morte celular por apoptose. Contudo, a proteina p53 e o supressor tumoral mais frequentemente mutado em tumores humanos, sendo este um dos mecanismos que confere resistencia a morte em celulas tumorais. Em geral, tumores HPV positivos, embora possam apresentar degradacao de p53 pela proteina viral E6, nao carregam mutacoes nesse gene e apresentam um melhor prognostico da doenca. Neste estudo buscamos comparar a expressao dos genes SERCA em tumores associados ao HPV e tumores carregando mutacao em TP53. Analises por microscopia confocal foram realizadas para avaliar a marcacao de SERCA (BODIPY-tapsigarsina) em uma linhagem celular termo-sensivel para mutacao de TP53 (selvagem – celulas cultivadas a 32°C; mutado – celulas cultivadas a 37°C). Alem disso, foram usados dados genomicos e clinicos do TCGA para tumores de orofaringe, tonsila e base de lingua (que incluem tanto tumores associado ao HPV quanto tumores associados a mutacao em TP53) comparando a expressao de mRNA dos tres genes SERCA (ATP2A1, ATP2A2 e ATP2A3). TP53 e ATP2A3 apresentaram significante correlacao de expressao (Spearman: 0,52; p=1.008e-5), sendo ambos os genes mais expressos nos tumores HPV positivos (p=1.981e-6). Como observado para TP53, tumores com maior expressao de ATP2A3 apresentaram tendencia a um melhor prognostico da doenca. Celulas selvagens para TP53 apresentaram uma marcacao dispersa de SERCA enquanto que celulas carregando mutacao em TP53 exibiram maior intensidade de SERCA proximo ao nucleo. Os dados revelam uma relacao inedita entre a expressao diferencial de isoformas de SERCA com alteracoes em p53, e sugerem a existencia de padroes de co-expressao associados com a infeccao por HPV como parte do mecanismo molecular da oncogenese viral e como possiveis biomarcadores dos pacientes acometidos com este tipo de câncer. Palavras-chave: Ca2+-ATPase. SERCA. TP53. HPV. Apoptose.

Boron (B) affects plasma membrane (PM) integrity and consequently modulates the P-type PM H+-ATPase activity creating a driving force for nutrient influx at the root level. Because citrus rootstocks respond differently to B supply, we hypothesised that PM H+-ATPase activity of varieties contrasting in horticultural traits would affect nutrient uptake by trees. Sweet orange (Citrus sinensis) trees grafted onto Rangpur lime (RL;Citrus limonia) or Swingle citrumelo (SW;Citrus paradisi x Poncirus trifoliata) were grown in nutrition solution with four B concentrations (0 [control concentration], 46, 230 and 460 mu M B, as H3BO3) up to 7 days of treatment imposition after plant adaptation into the hydroponic condition. SW exhibited higher B absorption, leaf B and enzyme activity than RL. The highest enzyme activity was achieved with 230 mu M of B 1 day after treatment imposition (ATI), whereas B excess impaired the PM H+-ATPase in all periods evaluated. Absorption of mineral nutrients correlated with PM H+-ATPase activity, with greater nutrient uptake per root unit in SW compared to RL. Leaf and root nutrient concentrations were equivalent to amounts absorbed and enzyme activity, with greater increments exhibited by trees grafted onto SW compared to RL. Effects of B supply on PM H+-ATPase activity explain distinct nutrient uptake patterns by trees, what supports fine-tuning fertilisation guidelines of citrus taking into account rootstock varieties.

Integrated signaling network involving abscisic acid (ABA), nitric oxide (NO), and indole-acetic acid (IAA) controls root morphogenesis during salt stress by a mechanism still poorly understood. The present data unveiled an ABA-NO-IAA interaction underlying radicular morphological responses to salinity. ThreeSolanum lycopersicumgenotypes were analyzed: wild type, ABA-insensitive mutant (sitiens), and auxin-responsive (DR5::GUS) plants. Nitric oxide fluorescence, nitrate reductase activity, auxin signaling, and some molecular analyses were performed. Pharmacological inhibitors and NO donor sodium nitroprusside were also used to evaluate NaCl-induced root morphological responses. Sodium nitroprusside inhibited primary root length, increased lateral root emergence, and rescued salt inhibited lateral root growth. The results showed that NO integrates the ABA-IAA signaling network of root system responses under salt stress, involving: (a) ABA and molybdenum-dependent enzymes as responsible for salt-induced NO production; (b) modulations of the plasma membrane H+-ATPase coupling and isoforms differential expression; and (c) ABA-mediated and NO-dependent antioxidative enzymes activities.

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

Background: Metastatic tumor cells have acidic extracellular pH and differential electrochemical 11 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 extra cellular 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 mu M 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.

ABSTRACT Attempts to improve the ascorbate (AsA) content of plants are still dealing with the limited understanding of why exists a wide variability of this powerful anti-oxidant molecule in different plant sources, species and environmental situations. In plant mitochondria, the last step of AsA synthesis is catalyzed by the enzyme L-galactone-1,4-lactone dehydrogenase (L-GalLDH). By using GalLDH-RNAi silencing plant lines, biochemical and proteomic approaches, we here discovered that, in addition to accumulate this antioxidant, mitochondria synthesize AsA to down-regulate the respiratory activity and the cellular energy provision. The work reveals that the AsA synthesis pathway within mitochondria is a branched electron transfer process that channels electrons towards the alternative oxidase, interfering with conventional electron transport. It was unexpectedly found that significant hydrogen peroxide is generated during AsA synthesis, which affects the AsA level. The induced AsA synthesis shows proteomic alterations of mitochondrial and extra-mitochondrial proteins related to oxidative and energetic metabolism. The most identified proteins were known components of plant responses to high light acclimation, programmed cell death, oxidative stress, senescence, cell expansion, iron and phosphorus starvation, different abiotic stress/pathogen attack responses and others. We propose that changing the electron flux associated with AsA synthesis might be part of a new mechanism by which the L-GalLDH enzyme would adapt plant mitochondria to fluctuating energy demands and redox status occurring under different physiological contexts.

This study tested the hypothesis that evaluating soil and leaf nutrient contents is useful to discriminate organic and conventional management systems of coffee plantations. The study consisted of areas planted with Coffea arabica under organic or conventional management located in Espirito Santo, Brazil. We detected significant differences between both management systems when we utilized multivariate statistics to discriminate the areas. In our experiments, the principal mineral leaf nutrients, which acted as indicators were phosphor (P), calcium (Ca), magnesium (Mg), sodium (Na), zinc (Zn), and manganese (Mn), with higher contents in the organic coffee production system. The only mineral nutrient, which showed significantly lower values in leaves as well as in soil of organic coffee is boron (B). The implications of these findings are discussed. However, when using univariate statistics, like ANOVA or t-test, we did not find any significant difference between both management systems, although using the same dataset. Therefore, to discriminate between complex systems, we always recommend to recur to multivariate methodologies that are more adequate for such cases.

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

V-ATPases are hetero-oligomeric enzymes consisting of 14 subunits and playing key roles in ion homeostasis and signaling. Differential expressions of these proton pumps have been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the V-ATPase genes expression in Esophageal Squamous Cell Carcinoma (ESCC) using gene expression microarray data and extended the analysis to other cancers the Oncomine database. Among all differentially expressed genes, those encoding the V-ATPase C isoforms exhibited striking expression patterns validated by qRT-PCR in paired ESCC samples and respective normal surrounding tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. This study reveals multi-cancer molecular signatures in the V-ATPase structure and establishes that the expression ratios of its subunits/isoforms could form a conformational code that controls the pump regulation and interactions related to tumorigenic events.