Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of ß-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Haloadaptative Responses of Aspergillus sydowii to Extreme Water Deprivation: Morphology, Compatible Solutes, and Oxidative Stress at NaCl Saturation.

Water activity (aw) is critical for microbial growth, as it is severely restricted at aw < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw ≅ 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.

Envelhecimento acelerado em sementes de alface / Accelerated aging in lettuce seeds

Objetivou-se adequar a metodologia do teste de envelhecimento acelerado para avaliação do potencial fisiológico de sementes de quatro lotes dos genótipos de alface 'Babá de verão', 'Tainá' e 'Vera', aplicando-se o método tradicional e com o uso de solução saturada de NaCl. Para tal, as sementes foram submetidas à determinação do teor de água, testes de germinação e vigor (primeira contagem, condutividade elétrica, emergência em bandeja, índice de velocidade de emergência) incluindo o teste de envelhecimento acelerado, conduzido nos períodos de 24, 48, e 72 horas, a 41°C com e sem solução saturada de NaCl. O experimento foi conduzido em delineamento inteiramente casualizado com quatro repetições. Os procedimentos tradicional e com o uso da solução saturada de NaCl por 72 horas foram sensíveis em estratificar os lotes em níveis de vigor, além de estarem de acordo com os demais testes utilizados. Portanto, o envelhecimento acelerado de sementes de alface deve ser feito utilizando o período de 72 horas, podendo ser realizado tanto pelo método tradicional quanto pelo método da solução saturada de NaCl.

The objective was to adapt the methodology of the accelerated aging test to evaluate the physiological potential of seeds of four lots of lettuce genotypes 'Babá de verão', 'Tainá' and 'Vera' applying the traditional method and using a saturated NaCl solution. So, the seeds were subjected to determination of water content, seed germination and vigor (first count, electrical conductivity, speed emergency index, emergency tray) including the accelerated aging test conducted in periods of 24, 48, 72 hours at 41°C with and without saturated solution of NaCl. The experiment was conducted in a randomized design with four replications. The accelerated aging traditional and saturated solution of NaCl by 72h was more sensitive to stratify the lots in vigor levels. Therefore, accelerated aging of seeds of lettuce should be made using the period of 72h and can be accomplished either by the traditional method and by the saturated NaCl solution method.