A multidisciplinary approach to the comparison of three contrasting treatments on both lampenflora community and underlying rock surface.

Removing lampenflora, phototrophic organisms developing on rock surfaces in tourist cavities due to the artificial lighting, is a challenge for sustainable and appropriate long-term management of caves. Photosynthetic-based biofilms usually cause rock biodeterioration and an ecological imbalance in cave ecosystems. In this work, a detailed investigation of the effects of the 3 most commonly used lampenflora cleaning operations (NaClO, H2O2 and UVC) was carried out in Pertosa-Auletta Cave (Italy). The application of NaClO showed good disinfection capability over extended periods of time without causing any appreciable rock deterioration. The H2O2 treatment showed to be corrosive for the rock surfaces covered with vermiculation deposits. The chemical alteration of organic and inorganic compounds by H2O2 did not remove biomass, favoring biofilm recovery after three months of treatment. Both NaClO and H2O2 treatments were effective at removing photoautotrophs, although the bacterial phyla Proteobacteria and Bacteroidetes as well as Apicomplexa and Cercozoa among the Eukaryotes, were found to be resistant to these treatments. The UVC treatments did not show any noticeable effect on the biofilms.

Microbial Community Characterizing Vermiculations from Karst Caves and Its Role in Their Formation.

The microbiota associated with vermiculations from karst caves is largely unknown. Vermiculations are enigmatic deposits forming worm-like patterns on cave walls all over the world. They represent a precious focus for geomicrobiological studies aimed at exploring both the microbial life of these ecosystems and the vermiculation genesis. This study comprises the first approach on the microbial communities thriving in Pertosa-Auletta Cave (southern Italy) vermiculations by next-generation sequencing. The most abundant phylum in vermiculations was Proteobacteria, followed by Acidobacteria > Actinobacteria > Nitrospirae > Firmicutes > Planctomycetes > Chloroflexi > Gemmatimonadetes > Bacteroidetes > Latescibacteria. Numerous less-represented taxonomic groups (< 1%), as well as unclassified ones, were also detected. From an ecological point of view, all the groups co-participate in the biogeochemical cycles in these underground environments, mediating oxidation-reduction reactions, promoting host rock dissolution and secondary mineral precipitation, and enriching the matrix in organic matter. Confocal laser scanning microscopy and field emission scanning electron microscopy brought evidence of a strong interaction between the biotic community and the abiotic matrix, supporting the role of microbial communities in the formation process of vermiculations.

Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake.

Stomatal movements rely on alterations in guard cell turgor. This requires massive K(+) bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K(+) into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K(+)/H(+) exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K(+) and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K(+) accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K(+) is a requirement for stomatal opening and a critical component in the overall K(+) homeostasis essential for stomatal closure, and suggest that vacuolar K(+) fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.

Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis.

Intracellular NHX proteins are Na(+),K(+)/H(+) antiporters involved in K(+) homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na(+),K(+)/H(+) antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K(+). By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na(+) sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K(+) pool, which in turn provoked greater K(+) retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K(+) uptake at the tonoplast, for turgor regulation, and for stomatal function.

Unveiling the menace of lampenflora to underground tourist environments.

Permanent artificial lighting systems in tourist underground environments promote the proliferation of photoautotrophic biofilms, commonly referred to as lampenflora, on damp rock and sediment surfaces. These green-colored biofilms play a key role in the alteration of native community biodiversity and the irreversible deterioration of colonized substrates. Comprehensive chemical or physical treatments to sustainably remove and control lampenflora are still lacking. This study employs an integrated approach to explore the biodiversity, eco-physiology and molecular composition of lampenflora from the Pertosa-Auletta Cave, in Italy. Reflectance analysis showed that photoautotrophic biofilms are able to absorb the totality of the visible spectrum, reflecting only the near-infrared light. This phenomenon results from the production of secondary pigments and the adaptability of these organisms to different metabolic regimes. The biofilm structure mainly comprises filamentous organisms intertwined with the underlying mineral layer, which promote structural alterations of the rock layer due to the biochemical attack of both prokaryotes (mostly represented by Brasilonema angustatum) and eukaryotes (Ephemerum spinulosum and Pseudostichococcus monallantoides), composing the community. Regardless of the corrosion processes, secondary CaCO3 minerals are also found in the biological matrix, which are probably biologically mediated. These findings provide valuable information for the sustainable control of lampenflora.

Connecting molecular biomarkers, mineralogical composition, and microbial diversity from Mars analog lava tubes.

Lanzarote (Canary Islands, Spain) is one of the best terrestrial analogs to Martian volcanology. Particularly, Lanzarote lava tubes may offer access to recognizably preserved chemical and morphological biosignatures valuable for astrobiology. By combining microbiological, mineralogical, and organic geochemistry tools, an in-depth characterization of speleothems and associated microbial communities in lava tubes of Lanzarote is provided. The aim is to untangle the underlying factors influencing microbial colonization in Earth's subsurface to gain insight into the possibility of similar subsurface microbial habitats on Mars and to identify biosignatures preserved in lava tubes unequivocally. The microbial communities with relevant representativeness comprise chemoorganotrophic, halophiles, and/or halotolerant bacteria that have evolved as a result of the surrounding oceanic environmental conditions. Many of these bacteria have a fundamental role in reshaping cave deposits due to their carbonatogenic ability, leaving behind an organic record that can provide evidence of past or present life. Based on functional profiling, we infer that Crossiella is involved in fluorapatite precipitation via urea hydrolysis and propose its Ca-rich precipitates as compelling biosignatures valuable for astrobiology. In this sense, analytical pyrolysis, stable isotope analysis, and chemometrics were conducted to characterize the complex organic fraction preserved in the speleothems and find relationships among organic families, microbial taxa, and precipitated minerals. We relate organic compounds with subsurface microbial taxa, showing that organic families drive the microbiota of Lanzarote lava tubes. Our data indicate that bacterial communities are important contributors to biomarker records in volcanic-hosted speleothems. Within them, the lipid fraction primarily consists of low molecular weight n-alkanes, α-alkenes, and branched-alkenes, providing further evidence that microorganisms serve as the origin of organic matter in these formations. The ongoing research in Lanzarote's lava tubes will help develop protocols, routines, and predictive models that could provide guidance on choosing locations and methodologies for searching potential biosignatures on Mars.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato.

NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na(+) in vacuoles. However, all isoforms characterized so far catalyze both Na(+)/H(+) and K(+)/H(+) exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K(+), which in turn affects plant K(+) nutrition and Na(+) tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K(+) vacuolar pools in all growth conditions tested, but no consistent enhancement of Na(+) accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K(+) and to withstand salt-shock. Under K(+)-limiting conditions, greater K(+) compartmentation in the vacuole occurred at the expense of the cytosolic K(+) pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K(+) uptake system, enhanced K(+) uptake by roots, and increased the K(+) content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H(+)-linked K(+) transport that is thought to facilitate active K(+) uptake at the tonoplast, and the partitioning of K(+) between vacuole and cytosol.

Lichen Vitality After a Space Flight on Board the EXPOSE-R2 Facility Outside the International Space Station: Results of the Biology and Mars Experiment.

As part of the Biology and Mars Experiment (BIOMEX; ILSRA 2009-0834), samples of the lichen Circinaria gyrosa were placed on the exposure platform EXPOSE-R2, on the International Space Station (ISS) and exposed to space and to a Mars-simulated environment for 18 months (2014-2016) to study: (1) resistance to space and Mars-like conditions and (2) biomarkers for use in future space missions (Exo-Mars). When the experiment returned (June 2016), initial analysis showed rapid recovery of photosystem II activity in the samples exposed exclusively to space vacuum and a Mars-like atmosphere. Significantly reduced recovery levels were observed in Sun-exposed samples, and electron and fluorescence microscopy (transmission electron microscope and field emission scanning electron microscope) data indicated that this was attributable to the combined effects of space radiation and space vacuum, as unirradiated samples exhibited less marked morphological changes compared with Sun-exposed samples. Polymerase chain reaction analyses confirmed that there was DNA damage in lichen exposed to harsh space and Mars-like environmental conditions, with ultraviolet radiation combined with space vacuum causing the most damage. These findings contribute to the characterization of space- and Mars-resistant organisms that are relevant to Mars habitability.

Cellular Responses of the Lichen Circinaria gyrosa in Mars-Like Conditions.

Lichens are extremely resistant organisms that colonize harsh climatic areas, some of them defined as "Mars-analog sites." There still remain many unsolved questions as to how lichens survive under such extreme conditions. Several studies have been performed to test the resistance of various lichen species under space and in simulated Mars-like conditions. The results led to the proposal that Circinaria gyrosa (Lecanoromycetes, Ascomycota) is one of the most durable astrobiological model lichens. However, although C. gyrosa has been exposed to Mars-like environmental conditions while in a latent state, it has not been exposed in its physiologically active mode. We hypothesize that the astrobiological test system "Circinaria gyrosa," could be able to be physiologically active and to survive under Mars-like conditions in a simulation chamber, based on previous studies performed at dessicated-dormant stage under simulated Mars-like conditions, that showed a complete recover of the PSII activity (Sánchez et al., 2012). Epifluorescence and confocal laser scanning microscopy (CLSM) showed that living algal cells were more abundant in samples exposed to niche conditions, which simulated the conditions in micro-fissures and micro-caves close to the surface that have limited scattered or time-dependent light exposure, than in samples exposed to full UV radiation. The medulla was not structurally affected, suggesting that the niche exposure conditions did not disturb the lichen thalli structure and morphology as revealed by field emission scanning electron microscopy (FESEM). In addition, changes in the lichen thalli chemical composition were determined by analytical pyrolysis. The chromatograms resulting from analytical pyrolysis at 500°C revealed that lichen samples exposed to niche conditions and full UV radiation consisted primarily of glycosidic compounds, lipids, and sterols, which are typical constituents of the cell walls. However, specific differences could be detected and used as markers of the UV-induced damage to the lichen membranes. Based on its viability responses after rehydration, our study shows that the test lichen survived the 30-day incubation in the Mars chamber particularly under niche conditions. However, the photobiont was not able to photosynthesize under the Mars-like conditions, which indicates that the surface of Mars is not a habitable place for C. gyrosa.

The Effect of High-Dose Ionizing Radiation on the Astrobiological Model Lichen Circinaria gyrosa.

The lichen Circinaria gyrosa is an astrobiological model defined by its high capacity of resistance to space conditions and to a simulated martian environment. Therefore, it became part of the currently operated BIOMEX experiment on board the International Space Station and the recent STARLIFE campaign to study the effects of four types of space-relevant ionizing radiation. The samples were irradiated with helium and iron ions at doses up to 2 kGy, with X-rays at doses up to 5 kGy and with γ rays at doses from 6 to 113 kGy. Results on C. gyrosa's resistance to simulated space ionizing radiation and its post-irradiation viability were obtained by (i) chlorophyll a fluorescence of photosystem II (PSII), (ii) epifluorescence microscopy, (iii) confocal laser scanning microscopy (CLSM), and (iv) field emission scanning electron microscopy (FESEM). Results of photosynthetic activity and epifluorescence show no significant changes up to a dose of 1 kGy (helium ions), 2 kGy (iron ions), 5 kGy (X-rays)-the maximum doses applied for those radiation qualities-as well as a dose of 6 kGy of γ irradiation, which was the lowest dose applied for this low linear energy transfer (LET) radiation. Significant damage in a dose-related manner was observed only at much higher doses of γ irradiation (up to 113 kGy). These data corroborate the findings of the parallel STARLIFE studies on the effects of ionizing radiation on the lichen Circinaria gyrosa, its isolated photobiont, and the lichen Xanthoria elegans. Key Words: Simulated space ionizing radiation-Gamma rays-Extremotolerance-Lichens-Circinaria gyrosa-Photosynthetic activity. Astrobiology 17, 145-153.

Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts.

Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative.

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing.

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.

Mutants of the Arabidopsis thaliana cation/H+ antiporter AtNHX1 conferring increased salt tolerance in yeast: the endosome/prevacuolar compartment is a target for salt toxicity.

Mutants of the plant cation/H(+) antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by (31)P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H(+) antiporter.

The phosphate transporter PHT4;6 is a determinant of salt tolerance that is localized to the Golgi apparatus of Arabidopsis.

Insertion mutations that disrupt the function of PHT4;6 (At5g44370) cause NaCl hypersensitivity of Arabidopsis seedlings that is characterized by reduced growth of the primary root, enhanced lateral branching, and swelling of root tips. Mutant phenotypes were exacerbated by sucrose, but not by equiosmolar concentrations of mannitol, and attenuated by low inorganic phosphate in the medium. Protein PHT4;6 belongs to the Major Facilitator Superfamily of permeases that shares significant sequence similarity to mammalian type-I Pi transporters and vesicular glutamate transporters, and is a member of the PHT4 family of putative intracellular phosphate transporters of plants. PHT4;6 localizes to the Golgi membrane and transport studies indicate that PHT4;6 facilitates the selective transport of Pi but not of chloride or inorganic anions. Phenotypic similarities with other mutants displaying root swelling suggest that PHT4;6 likely functions in protein N-glycosylation and cell wall biosynthesis, which are essential for salt tolerance. Together, our results indicate that PHT4;6 transports Pi out of the Golgi lumenal space for the re-cycling of the Pi released from glycosylation processes.

Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization.

Here, the function of the tomato (Solanum lycopersicon) K+/H+ antiporter LeNHX2 was studied using 35S-driven gene overexpression of a histagged LeNHX2 protein in Arabidopsis thaliana and LeNHX2 gene silencing in tomato. Transgenic A. thaliana plants expressed the histagged LeNHX2 both in shoots and in roots, as assayed by western blotting. Transitory expression of a green fluorescent protein (GFP) tagged protein showed that the antiporter is present in small vesicles. Internal membrane vesicles from transgenic plants displayed enhanced K+/H+ exchange activity, confirming the K+/H+ antiporter function of this enzyme. Transgenic A. thaliana plants overexpressing the histagged tomato antiporter LeNHX2 exhibited inhibited growth in the absence of K+ in the growth medium, but were more tolerant to high concentrations of Na+ than untransformed controls. When grown in the presence of NaCl, transgenic plants contained lower concentrations of intracellular Na+, but more K+, as compared with untransformed controls. Silencing of LeNHX2 in S. lycopersicon plants produced significant inhibition of plant growth and fruit and seed production as well as increased sensitivity to NaCl. The data indicate that regulation of K+ homeostasis by LeNHX2 is essential for normal plant growth and development, and plays an important role in the response to salt stress by improving K+ accumulation.

Transgenic tobacco plants overexpressing chitinases of fungal origin show enhanced resistance to biotic and abiotic stress agents.

Genes encoding defense-related proteins have been used to alter the resistance of plants to pathogens and other environmental challenges, but no single fungal gene overexpression has produced broad-spectrum stress resistance in transgenic lines. We have generated transgenic tobacco (Nicotiana tabacum) lines that overexpress the endochitinases CHIT33 and CHIT42 from the mycoparasitic fungus Trichoderma harzianum and have evaluated their tolerance to biotic and abiotic stress. Both CHIT33 and CHIT42, individually, conferred broad resistance to fungal and bacterial pathogens, salinity, and heavy metals. Such broad-range protective effects came off with no obvious detrimental effect on the growth of tobacco plants.

Alkali cation exchangers: roles in cellular homeostasis and stress tolerance.

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.

PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding.

The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG-N-CCGG inverted repeats and to CCGG-6/7N-CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG-6N-CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG-N-CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino-terminal to it. Three high-affinity sites in two different promoters have been visualized by in vivo methylation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high-affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiae protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.

Multiple GATA sites: protein binding and physiological relevance for the regulation of the proline transporter gene of Aspergillus nidulans.

In Aspergillus nidulans, proline can serve both as a carbon and a nitrogen source. The transcription of the prnB gene, encoding the proline transporter, is efficiently repressed only by the simultaneous presence of ammonium and glucose. Thus, repression of this gene demands the activation of the CreA repressor and the inactivation of the positive-acting GATA factor AreA. Repression of all other prn structural genes results largely from inducer exclusion. In an areA null mutation background, prnB is repressible by the sole presence of glucose. We have determined by EMSA and missing-base interference experiments that there are 15 AreA-binding sites in the prnD-prnB intergenic region. Only sites 13/14, in the proximity of the prnB TATA box, are clearly involved in transcriptional activation and regulation. Mutation of these sites mimics qualitatively the regulatory effect of an areA null mutation. The deletion of the TATA box has a measurable effect on the maximal level of prnB transcription but does not alter the regulation pattern of this gene.