Ms solid medium

Phenotypic analysis of sexual reproduction was conducted as described previously (Park et al., 2016 (link)). MATα and MATa isolates of WT and mutant strains were grown overnight in liquid YPD medium. Next, the cells were washed with water, mixed at equal amounts of cells (~1 × 10⁸ cells), spotted on MS solid medium (Sigma), and incubated in the dark at room temperature for 5–7 days. Images of sexual hyphae were captured using an Eclipse E400 microscope (Nikon) equipped with a DMX1200F camera (Nikon).
Pheromone gene expression was analyzed by culturing MATα and MATa cells of WT or mutant strains overnight. Next, the cultured cells were mixed at equal density, spotted on V8 solid medium (pH 5), and incubated in the dark at room temperature for 24 h. Samples were collected, frozen quickly, and stored at −80°C. Total RNA was isolated from each sample by using Trizol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions, and complementary DNA was synthesized using an AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies). Quantitative PCR was performed using the Brilliant III Ultra-Fast SYBR QPCR Mix (Agilent) and a StepOnePlus Real-Time PCR system (ABI). A housekeeping gene GPD1, encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), served as a control in the expression analysis.

For germination tests, seeds were sterilized in 70% (v/v) ethanol, 0.5% Triton X-100 (v/v) for 3 min and thereafter washed twice with 95% (v/v) ethanol. Seeds were sown on 50% MS solid medium (Sigma-Aldrich St Louis, MO, USA; M5524; Murashige and Skoog, 1962 ) on Petri dishes. To study the germination capacity under mitochondrial dysfunction stress, the MS medium was supplemented with 50 μM of AA. After 3 d at 4°C in darkness, the plates were transferred to 120 μmol photons m⁻² s⁻¹ at 8/16 h light/dark period under 23°C. The fresh weight of the seedlings was measured after 1 week of growth.
Stress treatments were performed on 4-week-old plants grown under control conditions of 120 μmol photons m⁻² s⁻¹ at 23°C and 50% relative humidity. UV-B stress was applied with Philips Broadband ultraviolet-B lamp (TL20W/12 RS; Philips, Amsterdam, Netherlands) at an irradiance of 1.5 W m m⁻² for 45 min. Thereafter, the plants were transferred to the control growth conditions, and samples were collected 24 h into the recovery period. AA was applied by spraying the rosettes with 50 μM AA in 0.01% (v/v) Tween-20, and samples were taken after a 10-h treatment under control growth conditions. For mock treatment, the plants were sprayed with 0.01% (v/v) Tween-20. The rosettes were harvested in pools of three, immediately frozen in liquid N₂ and stored at −80°C until protein isolation.