Imaging Singlet Oxygen Sensor Green fluorescence

Seedlings were grown for 2 d in the dark, followed by a 3‐d FR pretreatment. At 150 min before the end of the third day in FR, seedlings were immersed in a solution of 10 μM Singlet Oxygen Sensor Green (SOSG; ThermoFisher, Waltham, MA, USA) for 2 h in FR, then gently blotted dry and returned to their growth environment for 30 min. Seedlings were transferred to WL and excised cotyledons were imaged with fluorescence microscopy using a Zeiss Axioplan2 microscope (excitation = 470/40 nm; dichroic = 495 nm (LP); emission = 525/50 nm) with an integration time of 100 ms. Control seedlings either remained in the dark for 5 d or were imaged after FR without SOSG treatment to account for background fluorescence from the plant tissue. For the first time‐point (0 h, before transfer to WL), slides were prepared in the dark under a dim green safelight and maintained in the dark before imaging by wrapping in foil. All images were acquired using the same objective lens (×10), and intensity histograms were kept constant for all images shown. The SOSG signal for each sample was determined in Imagej (NIH, Bethesda, MD, USA) by assessing the signal averaged over the area of one cotyledon. Each data point represents the mean SOSG signal of three cotyledons from seedlings assayed in independent biological replicates. The same microscope settings were used to acquire all images.