Comparative analysis of nascent transcription among plant species
Overall design: A.thaliana Col-0 mature leaves were collected from plants grown as described (M. Wang et al. 2023), while A.thaliana Col-0 suspension cells were grown in 250-mL baffled flasks containing 50 mL of growth medium (3.2 g/L Gamborg’s B-5 medium, 3 mM MES, 3% [v/v] Suc, 1.1 mg L−1 2,4-dichlorophenoxyacetic acid). The cultures were maintained at 23°C under continuous light on a rotary shaker (160 rpm) and kindly provided as a frozen pellet by Dr. Ashley M. Brooks. Barley (Hordeum vulgare) RNA was isolated by Dr. Pete Hedley from embryonic tissue (including mesocotyl and seminal roots; EMB) isolated from grain tissues 4 days past germination (Mascher et al. 2017). Physcomitrium (Physcomitrella) patens (Gransden) was grown on plates with BCDA medium in a growth cabinet at 21°C under 16h light. Selaginella moellendorffii was purchased from Plant Delights Nursery and grown at the window under normal daylight for 1 week prior to isolating RNA from stems and leaves. C. reinhardtii, which was kindly provided by Dr. Will Ansari and Dr. Stephen Mayfield (UC San Diego), was grown to late logarithmic phase in TAP (Tris–acetate–phosphate) medium at 23°C under constant illumination of 5000 lux on a rotary shaker. Adult 2nd and 3rd leaves from Z. mays L. cultivar B73 was kindly provided by Dr. Lauri Smith (UC San Diego). Plants were grown in 4-inch pots in a greenhouse (temp: 23°C-29°C) without supplemental lighting or humidification (humidity in the 15 hours following inoculation ranged between 70 and 90%) year round in La Jolla, CA. RNA from Z. mays L. cultivar B73 7d old shoot, root and leaves were grown in the Schmitz laboratory (University of Georgia) as described in (Ricci et al. 2019).
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