Extended Data Fig. 5: NRT2.1 S501 phosphorylation acts as a negative phospho-switch that represses HATS activity.
From: A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1
a, Phenotypes of 14-day-old WT, nrt2.1-2.2 mutant, nrt2.1-2.2 complemented with NRT2.1[S501A] and nrt2.1-2.2 complemented with NRT2.1[S501D] grown on 1 mM NO3– medium. Scale bar = 2 mm. b, Fresh weight of 14-day-old shoots of the plants shown in (a). c, Root HATS activity of the plants shown in (a). d, Mass spectra of the NRT2.1 G478 nonphosphorylated peptide (position 478-493 in NRT2.1 protein) and ACT8 A21 peptide (position 21-30 in ACT8 protein) derived from WT and NRT2.1[S501D] plants. Decrease in ACT8 protein abundance in S501D plants suggests the decrease in overall protein expression level is due to a nitrate uptake defect in S501D plants. Considering this, protein abundance of NRT2.1 was not significantly affected by the S501D mutation. e, Root HATS activity of 14-day-old nrt2.1-2.2 mutant plants complemented with NRT2.1[S501A], ceph-1 mutant, ceph-1 mutant expressing NRT2.1[S501A] and ceph-1 mutant expressing NRT2.1[S501D]. The plotted S501A/nrt2.1-2.2 data is the same as that shown in Extended Data Fig. 5c, as the experiments were done concurrently. f, Mass spectra of the NRT2.1 G478 nonphosphorylated peptide (position 478-493 in NRT2.1 protein) derived from reciprocally 14N- and 15N-labeled plants, showing comparable NRT2.1 protein abundance in mock and estradiol-treated XVE-CEPH plants. Data are presented as the mean ± SD, and each dot represents a biological replicate. For (b), (c) and (e), different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with post-hoc Tukey’s test).
