Image used for GUS staining quantification in Hayes et al (2019) Current Biology: Figure 3

DR5v2:GUS plants in the Col or pif4-101/pif5 background were germinated for 3 days on soil (white light- 16:8h photoperiod), before being transferred to soil with or without 25mM NaCl. Plants were then grown in Wl for a further day. On day 4, at Zt 4.5, half the plants were moved to white light + far-red light. Tissues were fixed at Zt 4.5 on day 6 by 20 minutes in 90% acetone at -20°C. Tissues were washed twice under a vacuum in GUS wash solution (0.1M Phospho-PI pH 7.0, 10 mM EDTA, 2 mM K3Fe(CN)6) and then stained overnight at 37°C in GUS staining solution (0.1M Phospho-PI pH 7.0, 10 mM EDTA, 1 mM K3Fe(CN)6, 1mM K4Fe(CN)6 * 3H2O, 0.5 mg/ ml X-Gluc). The GUS reaction was inhibited by treatment with 3:1 ethanol: acetic acid mix for 1 hour at 37°C. Tissues were cleared with 70% ethanol over several days and then mounted in 10% chloral hydrate, 30% glycerol. Slides were scanned and the images used for quantification.

Sample order is as follows:

pif4pif5 Wl                                     Col Wl

pif4pif5 Wl                                     Col Wl

pif4pif5 Wl+ NaCl                          Col Wl+ NaCl

pif4pif5 Wl+ NaCl                          Col Wl+ NaCl

pif4pif5 Wl +FR                              Col Wl +FR         

pif4pif5 Wl +FR                              Col Wl +FR         

pif4pif5 Wl +NaCl +FR                  Col Wl +NaCl +FR

pif4pif5 Wl +NaCl +FR                  Col Wl +NaCl +FR