Fig. 4. Auxin regulates lateral root GSA through an RCN1-dependent PIN3 module.

a,b, Overexpression of RCN1 driven by the ARL2 promoter (ARL2::RCN1) in a WT Col-0 background results in a significantly more vertical lateral root GSA phenotype in contrast to Col-0 control (a) and restores the GSA of rcn1 lateral roots (b). n = 15–25 for each genotype from 3 biologically independent experiments for both a and b. Statistical analysis was performed using a two-tailed t-test for a and a one-way ANOVA with an F stat (2) = 19.3276 with P = 4.188 × 10⁻⁵. c,d, RCN1:GFP protein levels in 10-day-old lateral roots treated with 50 nM IAA for 4 h. Auxin treatment results in a significant increase in GFP signal in the columella cells of RCN1:GFP lateral roots. n = 15–21 roots per treatment from 3 biologically independent experiments. Statistical analysis was performed using a two tailed t-test (d). Red dashed lines represent columella area used for quantification (c). Scale bar, 20 μm (c). e,f, Visualisation (e) and quantification (f) of the effect of overexpression of RCN1 on PIN polarity in lateral root columella cells. RCN1 overexpression leads to a significant shift in PIN3:GFP polarity towards the lower side of the cell (d). In contrast, PIN7:GFP polarity is unaffected. n = 18–24 for each genotype from 3 biologically independent experiments. Statistical analysis was performed using a pairwise two-tailed t-test. Scale bar, 15 μm (e). g, The PP2A/RCN1 subunit is able to dephosphorylate the central hydrophilic loop of PIN3 in vitro. The experiment was repeated independently three times with similar results.