Measurements of 33P Uptake Applied as 33P-PO43- and 33P-ATP

For uptake measurement of ³³P-P_i (Hartmann Analytic, Braunschweig, Germany), roots were excised from P. x canescens plants, which were 14 to 18-weeks old and/or 0.7–1 m in height (Herschbach et al., 2010; Honsel et al., 2012). Roots of beech seedlings were excised after removing vermiculite and peat particles. Excised roots of both species were placed into an incubation chamber (Herschbach and Rennenberg, 1991), which consisted of three compartments, i.e., an application compartment (compartment A, 50 mL), a buffer compartment (compartment B, 20 mL) and a compartment for xylem sap exudation (compartment C, 30 mL). In case of poplar, for pre-incubation the compartments were filled with ¼ Hoagland solution (compartment B and C without P_i) supplemented with 2 mM MES buffer and adjusted to pH 5.0. In case of ³³P-ATP treatments, the respective (pre-) incubation solutions in compartment A did not contain phosphate and molybdate but ATP. Beech roots were pre-incubated in the beech fertilization solution supplemented with 2 mM MES buffer adjusted to pH 5.0. The pH dependency of P_i uptake was analyzed with excised poplar roots over a range of pH 3.5 to pH 7 (Hinsinger, 2001) and revealed highest values at pH 4.5 to pH 5.5, but no marked pH optimum (Supplementary Figure S1). Hence, all uptake experiments were performed at pH 5.0.

Incubation chambers were placed on aluminum plates cooled down to 15°C to simulate soil temperature. Excised roots of beech and poplar were pre-incubated for 2 h (Herschbach et al., 2010). After pre-incubation the solution of the application compartment (compartment A) was replaced by the respective solution supplemented with radiolabeled 0.25 mM ³³P-phosphate (4.1^∗10⁷ to 5.3^∗10⁷ Bq mmol^-1 P_i) or with 0.169 mM ³³P-ATP (5.3^∗10⁷ to 1.2^∗10⁷ Bq mmol^-1 ATP). ³³P-ATP was applied either as γ³³P-ATP or as α³³P-ATP (Figure 1). Uptake of ³³P from ³³P-P_i and ³³P-ATP was terminated after 4 h [during this time, linear uptake can be assumed (Herschbach and Rennenberg, 1991)] by washing the roots three-times with the respective unlabeled solution to remove adherent labeled compounds. Root sections of the incubation compartment were separated from the root part located in compartment B and C. ³³P was determined by liquid scintillation counting after sample bleaching as previously described (Herschbach et al., 2010; Scheerer et al., 2010). Calculation of uptake rates as well as of xylem loading rates was performed according to Herschbach and Rennenberg (1991).

Overview of the experimental designs with differently labeled ATP molecules. During the experiments, three differently labeled ATP molecules were applied: α³³P-ATP; γ³³P-ATP, ¹³C/¹⁵N labeled ATP (ATP ¹³C₁₀H₁₆¹⁵N₅O₁₃P₃ xNa) with the ¹³C label in the ribose and base. The base adenine/cytidine was additionally labeled by ¹⁵N. Molybdate was applied as a common acid phosphatase inhibitor to prevent cleavage of the γP_i and βP_i unit of the ATP molecule (Gallagher and Leonard, 1982; Cabello-Díaz et al., 2012). Uptake of nucleotides such as ATP, ADP, AMP and/or adenosine via yet unknown transporters is indicated. Unlabelled phosphate (P_i) in the solution competed with the P_i cleaved from ATP by extracellular phosphatases, ecto-apyrases, or nucleotidases (Wu et al., 2007; Riewe et al., 2008; Tanaka et al., 2014) for the uptake via P_i uptake transporters.