15n labeled nh4cl

For ¹⁵N uptake analysis at seedling stage, seedlings of WT and activation tagging mutants were cultured in the Yoshida nutrient solution for 7 days (Yoshida et al., 1976), except that 1.44 mm NH₄NO₃ was replaced by 2.88 mm NH₄Cl. Next, seedlings were treated with ¹⁵N‐labeled NH₄Cl (10% atom ¹⁵NH₄Cl; Sigma‐Aldrich). Roots and shoots were harvested at the time of 12, 24, 36 and 48 h after treatment and dried at 70°C.
To monitor ¹⁵N remobilization efficiency, seeds were germinated and cultured in the Yoshida solution containing ¹⁵N‐labeled 2.88 mm NH₄Cl (10% atom ¹⁵NH₄Cl; Sigma‐Aldrich) until the emergence of the seventh leaves (time T₀). At T₀, labeled root and leaf samples were harvested from five plants. The remaining plants were transferred to unlabeled nutrient solution and grown until the tenth leaf stage (time T₁; chase period). At T₁, roots, old leaves previously labelled (from first to seventh leaves) and newly emerged leaves (eighth to tenth leaves) were harvested from five plants. After drying and weighing, samples were ground and ¹⁵N abundance was estimated as described previously (Masclaux‐Daubresse and Chardon, 2011).

Seeds of WT, OX and osasn1 plants were germinated and seedlings were cultured for 7 d in Yoshida nutrient solution (Yoshida et al. 1976) , in which 1.44 mM NH₄NO₃ was replaced by 2.88 mM NH₄Cl. Next, seedlings were treated with ¹⁵N-labeled NH₄Cl (10% atom ¹⁵NH₄Cl; Sigma-Aldrich). Roots and shoots were harvested at 12, 24, 36 and 48 h after treatment and dried at 70°C. The dried samples were weighed and ground, and ¹⁵N abundance was estimated as described previously (Masclaux-Daubresse and Chardon 2011) (link).