Plant Materials and Treatments

Arabidopsis thaliana ecotype Columbia-0 (Col-0) is the genetic background for all wild type plants used in this work, except cue1-1 which is in the Bensheim (Be-0) ecotype background. Seed stocks pPIN1:PIN1-GFP, DR5:GUS, and cue1/nox1 were obtained from ABRC. Genetic crosses were performed using the cue1/nox1 and the noa1-1 mutant as donor in all the cases and the corresponding acceptor lines axr1-3, pin1, pPIN1:PIN1-GFP, and DR5:GUS, as described in Fernández-Marcos et al. (2011) and Sanz et al. (2014). axr1-3, axr6-3, and pin1 mutants were a kind gift from Dr. James A.H. Murray (Cardiff University, Cardiff, United Kingdom), nia1nia2noa1-2 mutant was a kind gift from Dr. José León (IBMCP-CSIC, Valencia, Spain), and venosa (ven) and dov1 mutants were kindly provided by Dr. José Luis Micol (UMH, Elche, Spain). axr1-3 mutant was generated by mutagenizing a Col-0 population with EMS (Lincoln et al., 1990), and it harbors a point mutation that changes a cysteine from the active site into an alanine, rendering the protein inactive. Arabidopsis plants used throughout this work were grown routinely in a growth chamber under 50–60% humidity, a temperature of 22°C, and with a 16 h light/8 h dark photoperiod at 80–100 μE m⁻² s⁻¹ in pots containing a 1:3 vermiculite:soil mixture.

For in vitro culture, Arabidopsis seeds were surface-sterilized in 75% (v/v) bleach solution (4–5% sodium hypochlorite) and 0.01% (v/v) Triton X-100 for 5 min and washed three times in sterile water before sowing. Seeds were stratified for 3 days at 4°C and then grown on MS medium (Murashige and Skoog, 1962). Petri dishes containing solid medium composed of MS basal salts and 2% (w/v) Gluc were solidified with 0.6% (w/v) agar, and the pH was adjusted to 5.7 with KOH before autoclaving. Plates were sealed and incubated horizontally in a controlled environment growth chamber. For the different treatments, seeds were sown in MS vertical sealed plates and maintain for 7, 10, 15, 21, and 30 days, after which were changed to vertical plates supplemented with 300 μM NO donor SNAP (S-Nitroso-N-acetyl-DL-penicillamine) or 1 mM NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] and incubated in the growth chamber during the different time periods.