Localization of the activity of the GDU promoters in Arabidopsis organs. GUS activity was revealed by histochemical staining of plants expressing GUS under the control of the GDU promoters. A, Root stele, GDU1 promoter. Similar staining was obtained for all other GDUs. B, Leaves, GDU5 promoter. Similar staining was obtained for GDU1, GDU2, and GDU3. C, Stem cross section, GDU3 promoter. Similar staining was obtained for GDU1 and GDU4. D, Flowers, GDU3 promoter.

gdu1-1D root growth is tolerant to exogenously supplied toxic amino acids. Wild-type (WT) and gdu1-1D (gdu) plants were grown vertically for 10 d on solid medium containing the amino acid indicated above each image (the concentration in mmol L⁻¹ is indicated in parentheses). Pro, Gln, Glu, Asn, Asp, and Ala had no effect on root growth; Lys, Trp, Cys, and Met inhibited equally the growth of wild-type and gdu1-1D roots; Val, Ser, Thr, Phe, Leu, Ile, His, Arg, and Gly inhibited strongly wild-type but not gdu1-1D root growth. [See online article for color version of this figure.]

Tolerance of the seven GDU overexpressors toward exogenously supplied amino acids. The GDU-OEs were grown vertically for 10 d on solid MS medium supplemented with 5 mm Ala, Asn, Asp, Gln, Glu, Gly, or Pro; 3 mm Ser; 2.5 mm Arg, Cys, His, Ile, Thr, or Val; 1 mm Phe; or 0.25 mm Leu or Lys. Means ± se of the length of five to 10 roots are shown. CTR, Plants expressing GFP under the control of the 35S promoter.

Amino acid export sensitivity to inhibitors of the proton gradient or ATP hydrolysis. Wild-type (WT) and gdu1-1D plants were pretreated with dimethyl sulfoxide (DMSO), 100 μm CCCP, 100 μm DNP, 1 mm VO₄, and 100 μm DES. The same amount of dimethyl sulfoxide was present in each sample. Uptake was performed for 10 min in the presence of 5 mm Phe; plants were allowed to release radioactivity for 10 min in the same medium without Phe. A, Total Phe taken up. DW, Dry weight. B, Percentage of Phe present in the medium after the efflux experiment, expressed as a percentage of the total Phe taken up. Means ± se of three biological replicates are shown. Significant differences from the wild type (t test) are as follows: * P < 0.05, ** P < 0.01.

Analysis of the Phe uptake and efflux by the GDU overexpressors. A, Seven-day-old plantlets were assayed for uptake of 1 mm Phe (light gray bars) or 0.1 mm Phe (dark gray bars) for 1 h. Phe accumulation is expressed as a percentage of the control (CTR) uptake (15.1 ± 1.1 and 2.9 ± 0.5 nmol mg⁻¹ dry weight for 1 and 0.1 mm Phe, respectively). Error bars represent the se of three biological replicates. *, Significantly different from the control (t test, P < 0.05). B, Analysis of Phe efflux for the GDU-overexpressing lines, performed as described in Table I. Amounts of Phe remaining in plants or released into the medium were calculated from the respective amounts of radioactivity. The number above each bar represents the percentage of incorporated radioactivity that was released into the medium. Error bars represent the se of three biological replicates. *, Percentage significantly different from the wild type (t test, P < 0.05). Uptake and efflux analyses were performed for two overexpressor lines per GDU gene with similar results, only one of which is presented for clarity and simplicity. CTR, Plants expressing GFP under the control of the 35S promoter.

Size and amino acid profiling of plants overexpressing the GDU genes. A, Rosettes of representative plants from one of the two overexpressing lines used in this study (Supplemental Table S1). The amount of secreted crystals at the margin of the leaves was lower for GDU2-, GDU3-, GDU5-, and GDU6-OEs than for GDU1-OEs. Bar = 1 cm. CTR, Plants expressing GFP under the control of the 35S promoter. B, One GDU-OE line per gene (Supplemental Table S1) was selected on kanamycin-containing medium for 7 d, transferred to soil, and grown for 3 weeks more. Leaves from eight plants were pooled and freeze dried. The amino acids were extracted and quantitated by HPLC. DW, Dry weight. Increases in the content of Pro, Gln, Ser, and Asn accounted for 50% to 65% of the total augmentation in free amino acid content, while His and Thr contents were increased the most (5- to 14-fold and 2- to 19-fold, respectively).

Analyses of the uptake of radiolabeled ¹⁴C-compounds by gdu1-1D and wild-type (WT) plants. A, Accumulation of Phe, Glu, His, methyl ammonium (MetNH₃⁺), and Glc (Gluc) after 10 min (supplied at 1 mmol L⁻¹) in whole plantlets. Wild type, light gray bars; gdu1-1D, dark gray bars. Means ± se of three biological replicates are shown. *, Significantly different from the wild type (t test, P < 0.02). DW, Dry weight. B, Accumulation of Phe, Arg, Pro, and Gly (supplied at 1 mmol L⁻¹) after 1 h in plants. Means ± se of three biological replicates are shown. *, Significantly different from the wild type (t test, P < 0.01). C, Concentration dependence of Phe uptake into wild-type plantlets treated with dimethyl sulfoxide (DMSO; black squares) or CCCP (100 μm; white diamonds). CCCP inhibits the proton gradient-dependent high-affinity uptake system and reveals the activity of the low-affinity uptake system. The difference between the uptake of the dimethyl sulfoxide- and the CCCP-treated plants corresponds to activity of the high-affinity amino acid uptake system, indicated by the broken line. Plants were allowed to take up Phe for 15 min before counting the amount of absorbed radioactivity. Means ± se of three biological replicates are shown. D, Time-course kinetics of the uptake of Phe supplied at a concentration of 50, 500, or 5,000 μmol L⁻¹ in wild-type (white squares) and gdu1-1D (black triangles) plants. Wild-type data points were fitted by the line. Means ± se of three biological replicates are shown. E, Effect of Phe pretreatment on time-course analysis of Phe uptake. Wild-type (squares) and gdu1-1D (triangles) plants were treated (dotted lines, white symbols) or not (solid lines, black symbols) with 1 mm Phe for 30 min prior to uptake analysis performed in the presence of 0.1 mm Phe. Means ± se of three biological replicates are shown.