Infrared-based growth rate and cotyledon movement analysis (IR Imaging):

To allow unobstructed visualization of hypocotyl and cotyledons in air, seedlings were grown vertically on the agar ledge formed by removing part of the agar in the square plate as previously described (Anwer et al., 2020). To image growth in day-night cycles we built an infrared imaging platform consisting of a modified camera with IR long pass 830 nm cut filters (Panasonic G5). Illumination was achieved using 880 nm IR backlights (Kingbright BL0106-15-29). Imaging was started at Zeitgeber time (ZT) 00 on day 3 (after germination). Photographs were taken every 60 min for 96 h in constant light (LL, white fluorescent lamps: 30 μmol m–2 s–1) under specified thermocycles (12 h 22 °C:12 h 16 °C or 12 h 28 °C:12 h 22 °C).  The imaging platform with infrared illumination was previously described (Anwer et al., 2020). Image stacks were analyzed using ImageJ (http://imagej.nih.gov/ij/). The circadian parameters of cotyledon movement were determined using the MFourFit method integrated in the BioDare2 analysis platform (Zielinski et al., 2014).