Quantification of R2D2, DII‐VENUS, and mDII‐VENUS fluorescence signal in Arabidopsis roots

R2D2 combines RPS5A‐driven DII (DII domain of the INDOLE‐3‐ACETIC ACID28 (IAA28, DII) from Arabidopsis) fused to n3×Venus and RPS5A‐driven mDII fused to ntdTomato on a single transgene (Brunoud et al, 2012; Liao et al, 2015). DII‐VENUS is the domain II of IAA28 fused to the VENUS fast maturing YFP, and mDII‐VENUS is the non‐degradable form of DII‐VENUS. The analysis of the fluorescence intensity of either R2D2, DII‐VENUS, or mDII‐VENUS expressing plants grown on ammonium containing and transferred on ammonium and nitrate‐containing medium was performed on Maximum Intensity Projection of Z‐stacks of root tips acquired with a Zeiss LSM 700 inverted laser‐scanning microscope as described in (Di Mambro et al, 2017) with slight modifications.

To quantify the fluorescence signal in each cell per selected root tissue (epidermis and cortex) first, we positioned a segmented line over the nuclei in the corresponding tissues with the ROI manager tool of the software Fiji (v1.52) (Appendix Fig S2C). Next, we analyzed the fluorescence plot profiles of the different lines with the peak analyzer function of the software Origin (OriginLab Corporation) to find local maxima along the lines, which represented the fluorescence value of the nuclei in the tissues. In case of R2D2, auxin distribution plots were derived by reciprocal mean values of the normalized n3xVenus/ntdTomato ratio. Relative auxin level data in each cell per tissue were graphed after data interpolation using the Origin built‐in algorithm for smoothing.