Figure 1.
Construction of a FRET reporter for glutamine and in vitro properties of the purified protein.
(A) Schematic design of the reporter. (B,C) FRET reporter with mutation D157N. (B) Kdapp = 6.9 mM Gln, dynamic range rmax−rmin = 2.21 (n = 3). (D) Specificity to all proteinogenic amino acids. Ratio change upon addition of 3 mM (black bars) or 10 mM (white bars) of the amino acids given in tree letter code (n = 3). (D,E) FRET reporter with mutations D157N and T70A. (D) Kdapp = 18.8 mM Gln, dynamic range rmax−rmin = 2.31 (n = 3). (E) Specificity and ratio change upon addition of 3 mM (black bar) or 10 mM (white bar) of the amino acids given in tree letter code (n = 3).
Figure 2.
In vivo function of the Gln (D157N) reporter in rdr6 plants.
(A) Confocal snapshots of a responding root tip (gray) with blue (CFP) and yellow (YFP) fluorescence. Higher magnification pictures are shown in the lower panels. (B) Normalized fluorescence ratio changes upon superfusion with Gln-containing nutrient solution of Arabidopsis rdr6 root tips. Numbers indicate Glutamine concentrations in mM. (C) Apparent saturation kinetics of the Ratio changes revealed a Kd of 8.5 mM.
Figure 3.
Similar Gln transport at pH 7.5 and partial reduction by a protonophore.
(A) Fluorescence changes that correspond to an influx of Gln at pH 7.5. Superfusion by Gln (in mM) is indicated by the black bars. (B) Fluorescence changes in the presence of the protonophore CCCP (100 µM). Gln is given at times marked with black bars. Numbers indicate Glutamine concentrations in mM. (C) Concentration dependence of the fluorescence. Half maximal ratio change was Kdapp = 8.2 mM (n = 2) at pH 7.5 (closed squares) and Kdapp = 17.9 at pH 5.5 in the presence of CCCP (100 µM, n = 3, closed circles). (D) Maximal ratio changes at pH 7.5 and pH 5.5 with CCCP relative to those at pH 5.5.
Figure 4.
Functional characteristics of CAT8 in yeast and oocytes.
(A) Uptake rates in 22Δ8AA yeast with 1 mM Gln. White bars: pH 5.5, black bars pH 7.5 (n = 4). The uptake by CAT8 was marginally significant at the p<0.05 level at pH 7.5 (*), using the Mann-Whitney test. (B) Uptake rates in oocytes at 1 mM Gln at pH 5.5 and pH 7.5. In the indicated experiments, Arg and Leu, or Glu and Phe, were added at 2 mM as competitors (n = 3).The uptake by CAT8 was significant at the p<0.05 level (Mann-Whitney test). (C) Concentration dependence of Gln uptake by CAT8. Uptake from water-injected oocytes was subtracted from CAT8-injected oocytes at pH 7.5. A linear background uptake was observed in both oocyte batches. (D) Current-voltage relations of CAT8-expressing oocytes in the absence (black circle) and presence (open triangle) of 1 mM Gln at pH 5.5 (n = 4, solid lines). The dashed line corresponds to the current-voltage relation of water-injected control oocytes from the same batch (closed triangles). Similar data were obtained at pH 7.5.
Figure 5.
Sub-cellular localization of CAT8 expressed from the endogenous promoter in the plasma membrane and the tonoplast.
(A–D) Subcellular localization of pCAT8::CAT8-GFP (A) in the root tip, (B) in the meristematic zone, (C) from the center to epidermal cells, (D) and guard cell. Scaling bars: 10 µm. (E) Ultrastructural analysis of pCAT8::CAT8-GFP plants with transmission electron microscopy and immunogold labeling using the GFP antibody. Tonoplast and plasma membrane localization of CAT8 is visible as black dots. The plasma membrane localization was highlighted by small arrows. Vacuole, v; plasmodesmata, pd; cell wall, cw. Scaling bar: 100 nm. (F) Immunogold labeling of CAT8-GFP in membranes of autophagocytotic structures (as). Scaling bar: 100 nm. (G, H) Quantitative analysis of the fluorescence intensity in plasma membranes and the tonoplast along the orange line. Fluorescence intensity histograms indicate similar fluorescence strength in the membranes.
Figure 6.
Localization of CAT8 to the plasma membrane and the tonoplast in the root tip.
35S::CAT8-GFP (A,D) and co-localization with FM4-64 (B,E, 15 min.) in the plasma membrane, but not in early endocytic vesicles, overlay (C,F). (G) Partial co-localization with FM4-64 (red, H) in 35S::CAT8-GFP plants after 2 h of BFA treatment. (I) Sub-cellular localization of 35S::GFP-CAT8 in the apical root zone. Scaling bars: 10 µm.
Figure 7.
Isolation and characterization of cat8-1 and 35S::CAT8 overexpressor lines.
(A) Schematic drawing of the position of the T-DNA insertion (black triangle) and the used primers in the cat8 gene. Verification of the T-DNA insertion on genomic DNA (left panel) and identification of homozygous plants by RT-PCR confirmed by amplification of a 1739 bp fragment (right panel). Actin served as control. In all cases, 30 cycles were used. (B) Growth on 1 µM MSX (upper panel) and 0.5 µM MSX (lower panel). From left to right: 35S::CAT8, wild type, cat8-1.