The pathway involving the paralogous transcription factors Rtg1 and Rtg3 was first described in Saccharomyces cerevisiae as the retrograde regulation that adapts cellular metabolism in response to the state of mitochondrial respiration. We investigated the evolution of this pathway by studying its target genes in respiratory-deficient mutants of Candida albicans—a phylogenetically distant and metabolically distinct yeast species. We show that in C. albicans the Rtg pathway is also responsible for adaptation to cellular stresses related to respiratory dysfunction, but the repertoire of its target genes is different than in S. cerevisiae, and includes genes encoding proteins involved in alternative respiration, oxidative stress, mitophagy, and other aspects of metabolism. We also traced the evolution of the main components of the Rtg pathway and its target genes in the budding yeast (Saccharomycotina) subphylum. We show that the system originated within this clade following a single duplication of the gene encoding the ancestor of Rtg1 and Rtg3, but employs other factors, like the regulatory proteins Rtg2 and Mks1 that were likely present in the last common ancestor of budding yeasts. The regulation of the Rtg transcription factors in C. albicans is different than in S. cerevisiae, as both Rtg2 and Mks1 were lost in the majority of Serinales. Among the target genes, of particular interest is the evolution of the alternative oxidase (Aox), which was either lost or duplicated in multiple independent events. The presence of Aox strongly correlates with the mitochondrially encoded Complex I—a major source of oxidative stress.

Cryptosporidiosis is a leading cause of diarrhea in young children and immunocompromised individuals, particularly AIDS/HIV patients. The only FDA-approved drug against cryptosporidiosis, nitazoxanide, has limited effectiveness in immunocompromised patients and is not approved for use in children under 1 year. Genomic analysis and previous studies proposed an alternative respiration pathway involving alternative oxidase (AOX) and type II NAD(P)H dehydrogenase (NDH2), which are thought to generate the mitosome membrane potential in Cryptosporidium parvum. Additionally, AOX was nominated as potential drug targets, based on its absence in mammalian hosts and sensitivity of parasite growth to known inhibitors of AOX. However, our study demonstrated that NDH2 is not localized in the mitosome, AOX is non-essential for parasite growth, and knockout lines lacking this enzyme are equally sensitive to AOX inhibitors. These findings indicate that AOX and NDH2 are not ideal candidates for future drug development against cryptosporidiosis and force a re-evaluation of models of how the mitosome generates its membrane potential.

Exploring key genes of the alternative respiration pathway, uncoupling, and calcium-binding proteins in methanol-treated rapeseed under drought stress

Candida albicans is a clinically significant fungal pathogen capable of adapting to diverse host environments, including steep oxygen gradients ranging from ~21% oxygen to anaerobic. The ability to withstand varied oxygen levels is paramount to establishing colonization and persisting in host niches, and oxygen deprivation can also augment antifungal resistance. In this study, we used RNA sequencing to compare the global transcriptomic profiles of two strains of C. albicans (SC5314 and CHN1) grown purely anaerobically to those grown aerobically. In C. albicans SC5314, we observed a strong induction of the alternative oxidase AOX2 and several genes encoding subunits of mitochondrial enzyme complexes I, II, and V, signifying a shift to alternative respiration. Consistent with the diminished ATP production from this process, there was a significant downregulation of genes associated with growth and metabolism, including histones and ribosomal proteins, as well as chitinases and other genes involved in cell wall remodeling. Interestingly, the anaerobic C. albicans cultures had decreased expression of candidalysin (ECE1) and other virulence factors, contrasting with other studies reporting enhanced pathogenicity under oxygen deprivation. There were a greater number of significantly upregulated genes in C. albicans CHN1 compared to SC5314; however, most of the top 50 upregulated genes under anaerobic conditions were consistent between the two strains. The predominant difference in down-regulated genes between the two strains could be mapped to differences in hyphal transformation under aerobic conditions. Overall, our study provides a window into the molecular mechanisms of C. albicans adaptation between aerobic to anaerobic environments.IMPORTANCECandida albicans is a leading cause of fungal infections in humans, posing significant clinical challenges due to its remarkable adaptability and increasing antifungal resistance. Anaerobic environments can promote antifungal resistance, necessitating a deeper understanding of how C. albicans adapts to anoxia. While much research has been done to identify mechanisms underlying adaptation to hypoxia (i.e., low oxygen), this is the first study evaluating the global transcriptomic response of C. albicans to anoxia (no oxygen). Here, we uncover key transcriptomic changes that enable C. albicans to survive in the absence of oxygen, which are distinct from those identified under hypoxic conditions. Our research addresses a gap in current knowledge that may be exploited for combatting antifungal resistance.

ABSTRACTLegumes are among the most utilised agronomic plant species due to their symbiotic association with N2‐fixing bacteria. Since N2 fixation entails high ATP cost, salt stress disrupts N2 fixation in the symbiont, but increases the production of osmolytes and antioxidant systems in the host plant. This results in competition for C allocation between osmoprotection in the host and continued supply to the symbiont for N acquisition, which may result in different plant responses to salinity. Two‐nodule types of plant species with contrasting carbon requirements for organic N2 fixation can be found within legume species; determinate and indeterminate. In this study, we tested responses of respiratory carbon metabolism, nitrogen assimilation and antioxidant machinery in leaves and roots of Phaseolus vulgaris (determinate nodules) and Pisum sativum (indeterminate nodules) 24 and 72 h after salt treatment (300 mM of NaCl). In P. sativum, we observed that nitrogenase activity was maintained at 24 h, but showed a strong decrease at 72 h together with cytochrome activity. On contrast, in P. vulgaris, respiration rates were maintained by an enhanced antioxidant activity under salinity although at the expense of nodule metabolism. Despite of the severity of the salt stress for N2 fixation, both species showed similar mechanisms to cope with salinity, like the maintenance of alternative respiration and increased antioxidant defence, that are worthy to be tested in the long term under field conditions.

Data on changes in the energy status and pro-/antioxidant metabolism activity at different stages of the dormancy and upon emergence from it in L. buds were obtained. A significant degree of water content (70–75%) and a low proportion of free water (50%) during the overwintering of buds were demonstrated. The freezing temperature of free water ranged from –6°C to –8°C, reflecting the high degree of meristematic tissues adaptation to low temperatures. During autumn-winter morphogenesis (from August to January), buds demonstrated stable rates of heat generation and O uptake, and a high proportion of cytochrome respiration (more than 70%). In January, compared to autumn, a significant increase in the rate and efficiency of energy storage and an increase in provxidant levels (the content of thiobarbituric acid reactive substances (TBARS) and HO content) were observed. During dormancy emergence in spring, an increase in rate of heat production and respiratory capacity, but a decrease in the energetically efficiency of respiration were observed. Compared to the autumn-winter period, the activity of energetically inefficient alternative respiration increased 4.5 times in spring, suggesting the involvement of alternative oxidase in maintaining pro-/antioxidant metabolism and plant adaptation to spring temperature fluctuations and increased insolation. In spring, compared to the dormant period, we observed a peak in provxidants accumulation and antioxidant enzymes activity. The maximal diversity and activity of SOD isoforms during spring morphogenesis may be related to the accumulation of HO in various cellular compartments, as a stable ROS and an important signaling molecule. We concluded that plants adapted to more favorable conditions do not exhibit the deep, organic dormancy which is characteristic of natural conditions. Energy metabolism parameters, the capacity and ratio of the cytochrome and alternative respiratory pathways, provxidants content and antioxidant enzymes activity can serve as physiological and biochemical markers of dormancy maintenance and emergence in buds.

Desiccation tolerance is a complex biological phenomenon that allows certain plants to survive extreme dehydration and revive upon rehydration. Although significant progress has been made in understanding the physiological and molecular mechanisms involved in desiccation tolerance, recovery mechanisms after prolonged desiccation periods are enigmatic. Combining physiological, biochemical, transcriptomic and metabolomic approaches, we investigated the role of prolonged desiccation on recovery of Selaginella bryopteris. Prolonged desiccation causes a decline in the antioxidant system, leading to accumulation of ROS that hinder recovery by inducing cellular damage. Transcriptome and WGCNA analysis revealed the significance of protective proteins, alternative respiration and protein homeostasis in cellular protection and recovery after short and long-term desiccation. Metabolomic analysis exhibited an increased accumulation of antioxidant compounds, which can be substituted for antioxidant enzymes to maintain cellular protection during prolonged desiccation. The significant role of autophagy and autophagic components was evaluated by H2O2 treatment and phylogenetic analysis of ATG4 and ATG8, which unveiled their substantial role in desiccation tolerance and remarkable conservation of the autophagy-related genes across plant species. Our data demonstrated that prolonged desiccation leads to ROS-induced cell death by extensive autophagy due to enormous loss of protective proteins, antioxidant enzymes and energy resources during desiccation.

Cryptosporidium parvum and C. hominis possess a remanent mitochondrion called the mitosome, which lacks DNA, the tricarboxylic acid cycle, a conventional electron transport chain, and ATP synthesis. The mitosome retains ubiquinone and iron sulfur cluster biosynthesis pathways, both of which require protein import that relies on the membrane potential. It was previously proposed that the membrane potential is generated by electrons transferred through an alternative respiratory pathway coupled to a transhydrogenase (TH) that pumps hydrogens out of the mitosome. This pathway relies on an alternative oxidase (AOX) and type II NADH dehydrogenase (NDH2), which also exists in plants, some fungi, and several protozoan parasites. To examine this model, we determined the location and function of AOX and NDH2 in C. parvum. Surprisingly, we observed that NDH2 was localized to parasite surface membranes instead of the mitosome. Furthermore, a Δndh2 knockout (KO) strain was readily obtained, indicating that this protein is not essential for parasite growth. Although, AOX exhibited a mitosome-like staining pattern, we readily obtained an Δaox knockout strain, indicating that AOX is also dispensable for parasite growth. The growth of the Δaox strain was inhibited by the AOX inhibitors SHAM and 8-HQ to the same extent as wild type, indicating that AOX is not the target of these inhibitors in C. parvum. Collectively, our studies indicate that NDH2 and AOX are non-essential genes in C. parvum, necessitating an alternative mechanism for maintaining the mitosome membrane potential.ImportanceCryptosporidiosis is the leading cause of diarrhea in young children and immunocompromised individuals, particularly AIDS/HIV patients. The only FDA approved drug against cryptosporidiosis, nitazoxanide, has limited effectivity in immunocompromised patients and is not approved for usage in children under 1 year old. Genomic analysis and previous studies proposed an alternative respiration pathway involving alternative oxidase (AOX) and type II NAD(P)H dehydrogenase (NDH2), which are thought to generate the mitosome membrane potential in C. parvum. Additionally, AOX and NDH2 were nominated as potential drug targets, based on their absence in mammalian hosts and sensitivity of parasite growth to known inhibitors of AOX. However, our study demonstrated that NDH2 is not localized in mitosome, AOX non-essential for parasite growth, and knockout lines lacking this enzyme are equally sensitive to AOX inhibitors. These findings indicate that AOX and NDH2 are not ideal candidates for future drug development against cryptosporidiosis and force a re-evaluation for models of how the mitosome generate its membrane potential.

Artificial electron snorkels reduce CH4 emissions in paddy soil: Regulation of the electron transfer pathway and microbial community ecology

Revisiting the intron hypothesis of QoI resistance in <i>Phyllosticta ampelicida</i>, the causal agent of grape black rot, and other <i>Phyllosticta</i> species

Brassinosteroids (BRs) represent a group of plant signaling molecules with a steroidal skeleton that play an essential role in plant adaptation to different environmental stresses, including drought. In this work, the effect of pretreatment with 0.4 µM 24-epibrassinolide (EBR) on the oxidant/antioxidant system in 4-day-old wheat seedlings (Triticum aestivum L.) was studied under moderate drought stress simulated by 12% polyethylene glycol 6000 (PEG). It was revealed that EBR-pretreatment had a protective effect on wheat plants as evidenced by the maintenance of their growth rate, as well as the reduction in lipid peroxidation and electrolyte leakage from plant tissues under drought conditions. This effect was likely due to the ability of EBR to reduce the stress-induced accumulation of reactive oxygen species (ROS) and modulate the activity of antioxidant enzymes. Meanwhile, EBR pretreatment enhanced proline accumulation and increased the barrier properties of the cell walls in seedlings by accelerating the lignin deposition. Moreover, the ability of EBR to prevent a drought-caused increase in the intensity of the total dark respiration and the capacity of alternative respiration contributes significantly to the antistress action of this hormone.

BACKGROUND: Plants as sessile organisms have developed biochemical pathways to protect themselves from the excess light energy. Mitochondrial alternative oxidase (AOX) participates in the oxidation of reductants exported from chloroplasts, thereby optimizing photosynthesis and protecting cells from photodamage.&#x0D; AIM: The effect of high light on respiration and the relative transcripts content of a number of genes in Arabidopsis thaliana plants of the T-DNA insertional line for AOX1a (aox1a) was studied and compared with the response of the antisense silencing of AOX1a line (AS-12) and wild type line Col-0.&#x0D; MATERIALS AND METHODS: Four-week-old A. thaliana plants of three lines grown at 90 µmol/m2 · s and then exposed to moderately high light conditions, 400 µmol/m2 · s, in a short-term experiment (8 h). Respiratory pathways activity, gene expression, and superoxide anion content were determined during experiment.&#x0D; RESULTS: Plants of the aox1a line in response to high light were characterized by the absence of the total and alternative respiration reaction and the absence of the AOX1 protein in spite of the increased mRNA level of AOX1c, in contrast to the Col-0 and AS-12 lines. Also, an increased content of transcripts of only SAPX and CHS were found, while in the other lines a compensatory increase in the expression of many “defense” genes was revealed.&#x0D; CONCLUSIONS: Thus, the aox1a line was characterized by a low compensatory effect at the level of defense systems activation. This is apparently caused by the absence of the AOX1 protein and, as a result, the weakening of the stress signal and stress response. The results obtained indicate the important role of AOX in the response of respiration to light stress; can be used to study the signaling pathways of regulation of AOX1a expression.

Commercial formulations of beneficial microbes have been used to enrich the rhizosphere microbiome of tomato plants grown in pots located in a glasshouse. These plants have been subjected to attacks by soil-borne parasites, such as root-knot nematodes (RKNs), and herbivores, such as the miner insect Tuta absoluta. The development of both parasites and the symptoms of their parasitism were restricted in these plants with respect to plants left untreated. A mixture, named in the text as Myco, containing plant growth-promoting rhizobacteria (PGPR), opportunistic biocontrol fungi (BCF), and arbuscular mycorrhizal fungi (AMF) was more effective in limiting pest damage than a formulation containing the sole AMF (Ozor). Therefore, Myco-treated plants inoculated with RKNs were taken as a model for further studies. The PGPR contained in Myco were not able to reduce nematode infection; rather, they worsened symptoms in plants compared with those observed in untreated plants. Therefore, it was argued that both BCF and AMF were the microorganisms that colonized roots and stimulated the plant immune system against RKNs. Beneficial fungi colonized the roots by lowering the activities of the defense supporting enzymes endochitinases and β-1,3-glucanase. However, as early as three days after nematode inoculation, these enzyme activities and the expression of the encoding pathogenesis-related genes (PR-2, PR-3) were found to be enhanced in roots with respect to non-inoculated plants, thus indicating that plants had been primed against RKNs. The addition of paclobutrazol, which reduces salicylic acid (SA) levels in cells, and diphenyliodonium chloride, which inhibits superoxide generation, completely abolished the repressive effect of Myco on nematode infection. Inhibitors of copper enzymes and the alternative cyanide-resistant respiration did not significantly alter resistance induction by Myco. When Myco-treated plants were subjected to moderate water stress and inoculated with nematodes, they retained numbers of developed individuals in the roots similar to those present in regularly watered plants, in contrast to what occurred in roots of untreated stressed plants that hosted very few individuals because of poor nutrient availability.

Transcriptome profiles reveal NF-YC1-regulated pathways in adaption to salt stress in Arabidopsis thaliana

Novel lines of tobacco (Nicotiana tabacum L.), highly expressing the AtNDB2 gene (NDB2 from Arabidopsis thaliana (L.) Heynh.), were produced with the help of agrobacterial transformation followed by a selection. The transgenic 13s line, possessing typical exterior and the AtNDB2 expression level, was compared with the initial wild type of N. tabacum regarding the parameters of growth and respiratory activity under optimal and suboptimal temperatures. It was found that the total and alternative respiration increased and the superoxide anion generation decreased in the 13s plants under the suboptimal temperature. The growth rate was decreased in the plants highly expressing the AtNDB2 gene in comparison with the control wild type plants, especially at the temperature below the optimum. Possible causes of the found changes are discussed.

The growth and persistence of alfalfa (Medicago sativa), a perennial legume capable of producing high yields of high-quality forage, is reduced in moderately acidic soils. The low performance of alfalfa at low pH is due to numerous factors that affect the host plant, their rhizobia, and the symbiotic interaction. Sinorhizobium meliloti LPU63 was isolated from acid topsoil (in Argentina) and showed to be a highly competitive and efficient N2-fixing rhizobium under both neutral and moderately acidic soil conditions. In this study, we obtained a draft of the LPU63 genome sequence using Illumina HiSeq4000. The whole genome phylogenetic analysis confirmed the taxonomic position of LPU63 as a S. meliloti strain and the multilocus sequence analysis confirmed that LPU63 is not related to the strains used in Argentina in bioformulations. The genomic analysis showed that beyond the canonical chromosome, pSymA, and pSymB, LPU63 strain has an accessory plasmid that codes for a repABC origin of replication and a conjugative T4SS, suggesting that this plasmid could be self-transmissible. In addition, the complete denitrification pathway (i.e., the gene clusters nap, nir, nor, and nos), including napC and nosZ, which could be used as an alternative respiration route under hypoxic conditions with moderate N2O emissions was found. Also, genes associated with plant growth-promoting activities (PGPR) and the degradation of phenylacetic acid (PAA) were identified. LPU63 is a highly melanogenic strain, a property that could enhance its survival under soil conditions, and the genome data showed a particular arrangement of the genes involved in melanin production. The information regarding LPU63 activities compatible with plant-growth promotion phenotypes, together with other characteristics mentioned here (melanin production, potential moderate N2O emissions), constitute the basis of future experiments toward the rational design of a novel bioinoculant for the environmentally sustainable production of alfalfa.

Recurring heat and drought episodes present challenges to the sustainability of grape production worldwide. We investigated the impacts of heat and drought stress on transcriptomic and metabolic responses of berries from two wine grape varieties. Cabernet Sauvignon and Riesling grapevines were subjected to one of four treatments during early fruit ripening: (1) drought stress only, (2) heat stress only, (3) simultaneous drought and heat stress, (4) no drought or heat stress (control). Berry metabolites, especially organic acids, were analyzed, and time-course transcriptome analysis was performed on samples before, during, and after the stress episode. Both alone and in conjunction with water stress, heat stress had a much more significant impact on berry organic acid content, pH, and titratable acidity than water stress. This observation contrasts with previous reports for leaves, which responded more strongly to water stress, indicating that grape berries display a distinct, organ-specific response to environmental stresses. Consistent with the metabolic changes, the global transcriptomic analysis revealed that heat stress had a more significant impact on gene expression in grape berries than water stress in both varieties. The differentially expressed genes were those associated with the tricarboxylic acid cycle and glyoxylate cycle, mitochondrial electron transport and alternative respiration, glycolysis and gluconeogenesis, carbohydrate allocation, ascorbate metabolism, and abiotic stress signaling pathways. Knowledge regarding how environmental stresses, alone and in combination, impact the berry metabolism of different grape varieties will form the basis for developing recommendations for climate change mitigation strategies and genetic improvement.

Profiling proteomic responses to hexokinase-II depletion in terpene-producing Saccharomyces cerevisiae

Haloferax mediterranei is the model microorganism for the study of the nitrogen cycle in haloarchaea. This archaeon not only assimilate N-species such as nitrate, nitrite, or ammonia, but also it can perform denitrification under low oxygen conditions, using nitrate or nitrite as alternative electron acceptors. However, the information currently available on the regulation of this alternative respiration in this kind of microorganism is scarce. Therefore, in this research, the study of haloarchaeal denitrification using H. mediterranei has been addressed by analyzing the promoter regions of the four main genes of denitrification (narGH, nirK, nor, and nosZ) through bioinformatics, reporter gene assays under oxic and anoxic conditions and by site-directed mutagenesis of the promoter regions. The results have shown that these four promoter regions share a common semi-palindromic motif that plays a role in the control of the expression levels of nor and nosZ (and probably nirK) genes. Regarding the regulation of the genes under study, it has been concluded that nirK, nor, and nosZ genes share some expression patterns, and therefore their transcription could be under the control of the same regulator whereas nar operon expression displays differences, such as the activation by dimethyl sulfoxide with respect to the expression in the absence of an electron acceptor, which is almost null under anoxic conditions. Finally, the study with different electron acceptors demonstrated that this haloarchaea does not need complete anoxia to perform denitrification. Oxygen concentrations around 100 μM trigger the activation of the four promoters. However, a low oxygen concentration per se is not a strong signal to activate the promoters of the main genes involved in this pathway; high activation also requires the presence of nitrate or nitrite as final electron acceptors.

Mitochondrial alternative oxidase enhanced ABA-mediated drought tolerance in Solanum lycopersicum