Extended Data Fig. 2: PIN mediated IAA and 2,4-D export from Xenopus oocytes and basipetal auxin transport in inflorescence Arabidopsis stems.
From: Transport of phenoxyacetic acid herbicides by PIN-FORMED auxin transporters
a) Time course of [3H]-IAA export by PIN8 from oocytes. Data of a typical experiment. Relative IAA content of oocytes expressing PIN8 in the presence (▲) or absence (•) of 10 μM NPA internally determined at the time indicated after substrate injection. Initial internal IAA concentration was 1 μM. n = 10 oocytes at each time point. Data points are mean ± SE. b) Relative transport rates of [3H]-IAA export by PIN8 in presence (dark grey) or absence (light grey) of 10 μM NPA as indicted from oocytes calculated from three independent time course experiments. Data points are individual experiments and bars represent mean ± SD. Groups were compared by a two-sided unpaired Student’s t-test; *** P < 0.0001. c) Time course of [3H]-2,4-D export by PIN8 from oocytes. Data of a typical experiment. Relative IAA content of oocytes expressing PIN8 in the presence (■) or absence (•) of 10 μM NPA internally determined at the time indicated after substrate injection. Initial internal 2,4-D concentration was 1 μM. n = 10 oocytes at each time point. Data points are mean ± SE. d) Time course of [3H]-IAA export by PIN3 from oocytes. Data of a typical experiment. Relative IAA content of oocytes expressing PIN3 without kinase in the presence (■) or absence (•) of 10 μM NPA internally and PID in the presence (▼) or absence (▲) of 10 μM NPA internally determined at the time indicated after substrate injection. Initial internal IAA concentration was 1 μM. n = 10 oocytes at each time point. Data points are mean ± SE. e) Relative transport rates of [3H]-IAA export by PIN3 with or without PID in presence (dark grey) or absence (light grey) of 10 μM NPA as indicated from oocytes calculated from three independent time course experiments. Data points are individual experiments and bars represent mean ± SD. Groups were compared by a two-sided unpaired Student’s t-test. n.s. not significant (P =0.99), *** P < 0.0001. f) Time course of [3H]-2,4-D export by PIN3 from oocytes. Data of a typical experiment. Relative 2,4-D content of oocytes expressing PIN3 without kinase in the presence (■) or absence (•) of 10 μM NPA internally and PID in the presence (▼) or absence (▲) of 10 μM NPA internally determined at the time indicated after substrate injection. Initial internal 2,4-D concentration was 1 μM. n = 10 oocytes at each time point. Data points are mean ± SE. g) Illustration of the stem transport assay measured in inflorescence stems of 5-week-old Arabidopsis plants. 2 cm stem sections were cut above the rosette of 5-week-old plants and placed in an inverted orientation into auxin transport buffer containing [3H]-IAA or [3H]-2,4-D as a substrate in the absence and presence of NPA. After two hours, 5 mm segments were dissected, and the substrate was quantified using a liquid scintillation counter. Segment 1 represents 0.5 to 1 cm, Segment 2 represents 1 to 1.5 cm and Segment 3 represents 1.5 to 2 cm of the stem, from apical to basal position. h) [3H]-IAA content in segments of Col-0 wildtype and pin1 mutant plants in the absence and presence of NPA as indicated. We used n = 5 individual inflorescences for segment analyses. Data points are segments of individual plant stems and bars represent mean ± SD. Groups were compared by a two-sided unpaired Student’s t-test. For individual p-values see Supplementary Table 2. i) [3H]-2,4-D content in segments of Col-0 wildtype and pin1 mutant plants in the absence and presence of NPA as indicated. We used n = 5 segments of individual inflorescences for Col-0 segments 2 and 3 without NPA, Col-0 segments 1, 2 and 3 with NPA, pin1 segments 1, 2 and 3 without NPA and pin1 segments 2 and 3 with NPA and n = 4 stem segments of individual inflorescences for Col-0 segment 1 without NPA and pin1 segment 1 with NPA. Data points are segments of individual plant stems and bars represent mean ± SD. Groups were compared by a two-sided unpaired Student’s t-test. For individual p-values see Supplementary Table 2.
