Constitutive Defense Mechanisms Have a Major Role in the Resistance of Woodland Strawberry Leaves Against Botrytis cinerea.

The necrotrophic fungus Botrytis cinerea is a major threat to strawberry cultivation worldwide. By screening different Fragaria vesca genotypes for susceptibility to B. cinerea, we identified two genotypes with different resistance levels, a susceptible genotype F. vesca ssp. vesca Tenno 3 (T3) and a moderately resistant genotype F. vesca ssp. vesca Kreuzkogel 1 (K1). These two genotypes were used to identify the molecular basis for the increased resistance of K1 compared to T3. Fungal DNA quantification and microscopic observation of fungal growth in woodland strawberry leaves confirmed that the growth of B. cinerea was restricted during early stages of infection in K1 compared to T3. Gene expression analysis in both genotypes upon B. cinerea inoculation suggested that the restricted growth of B. cinerea was rather due to the constitutive resistance mechanisms of K1 instead of the induction of defense responses. Furthermore, we observed that the amount of total phenolics, total flavonoids, glucose, galactose, citric acid and ascorbic acid correlated positively with higher resistance, while H2O2 and sucrose correlated negatively. Therefore, we propose that K1 leaves are more resistant against B. cinerea compared to T3 leaves, prior to B. cinerea inoculation, due to a lower amount of innate H2O2, which is attributed to a higher level of antioxidants and antioxidant enzymes in K1. To conclude, this study provides important insights into the resistance mechanisms against B. cinerea, which highly depend on the innate antioxidative profile and specialized metabolites of woodland strawberry leaves.

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 µM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography-mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.