FIG 1.

Copper stress regulates the accumulation of intracellular copper, hyphal branching, and GA biosynthesis in G. lucidum. (A) The G. lucidum WT strain was cultured in liquid CYM with shaking at 28°C. On the 4th day, various concentrations of copper were added. On the 7th day, the samples were analyzed. The levels of intracellular copper accumulation were measured by microwave digestion generation atomic fluorescence spectrometry and ICP-OES. (B) Effect of various copper concentrations on hyphal growth and colony diameter. (C) A UPLC method was used to measure the GA content in G. lucidum cultures treated with various concentrations of copper. (D to F) Measurements of the relative expression of genes that encode key enzymes in the GA biosynthetic pathway under various concentrations of copper: hmgr (encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase), sqs (encoding squalene synthase), and osc (encoding lanosterol synthase). (G) The G. lucidum WT strain was cultured on CYM plates for 5 days with various concentrations of copper. Aerial hyphae were removed from actively growing colonies, suspended in 2.5 μg · ml⁻¹ Fluorescent brightener 28, and observed using a Nikon Eclipse Ti-S microscope with UV light. The distance between two hyphal branches was measured in the fluorescence image. (H) Changes in the GA contents in copper-stressed cultures in the presence of a copper chelator. (I) Changes in the hyphal branch distance in G. lucidum in copper-stressed cultures in the presence of a copper chelator. All values are the mean ± standard deviation (SD) of the results from three independent experiments. Within each set of experiments, different letters indicate significant differences between the lines (P < 0.05, according to a multiple-comparison one-way ANOVA).