Figure 1.
Primary root growth is inhibited by ammonium in Arabidopsis thaliana.
A. thaliana Columbia plants were precultured on basal medium with 1 mM KNO3 as the sole nitrogen source, and then seedlings with 2 cm primary roots were transferred to a vertical two-layer split plate system with 1 mM nitrate in the upper layer and either 1 mM nitrate or ammonium in the lower layer, thus only apical portion of the root system contacted the treatment medium. (A) Photographs of representative seedlings in nitrate and ammonium treatment at five days after treatment. Bar = 1 cm. (B) Primary root length. Data are means ± SE (n = 4) from four replicate plates, with 4 seedlings each. Means for nitrate and ammonium treatment are significantly different: * p<0.05; ** p<0.01; *** p<0.001; ns = not significant. (C) Shoot fresh weight. Data are means ± SE (n = 4) from four replicate plates, with 4 seedlings each. (D) Primary root length in response to different forms of nitrogen. Data represent means ± SE (n = 4) from four replicate plates, with 4 seedlings each. Different letters indicate significant differences at p<0.05. (E) Primary root growth rate with the indicated composition of in the lower layer. Data represent means ± SE (n = 4) from four replicate plates, with 4 seedlings each.
Figure 2.
Kinematic analysis of root growth in response to ammonium treatment.
Seedlings were cultured as for Figure 1A and data were obtained at five days after treatment. For (A) cell length, cortical cells were measured. Profiles of (B) velocity and (C) elemental elongation rate were obtained as described in the methods. Data plot means ± SE (n = 6 to 7).
Table 1.
Effects of ammonium on primary root growth.
Figure 3.
Ammonium decreases apparent root meristem size and the number of dividing cells.
(A) Micrographs of PI-stained primary root tips at five days after treatment. Images are representative of 16 to 20 plants in four replicate experiments, treated as in Figure 1A. White arrowheads indicate the approximate position where cells begin to elongate noticeably. Bar = 50 µm. (B) Apparent root meristem length and (C) number of meristematic cortex cells (per file), seedlings treated as in Figure 1A. Bars plot means ± SD for 15 to 16 plants obtained in four replicate experiments. *** p<0.001. (D) Micrographs of the cell cycle reporter CycB1;1::GUS taken at the indicated days after transfer (DAT). Plants were treated as in Figure 1A. Bar = 50 µm. (E) The number of dividing cells as determined by counting the blue-green puncta in CycB1;1::GUS stained root tips. Bars plot means ± SD for 12 to 16 plants obtained in four replicate experiments. Different letters indicate significant differences at p<0.05.
Figure 4.
Ammonium does not alter the root stem cell activity, but reduced columella cell number.
(A) Confocal fluorescence micrographs of PI-stained root tips taken at five days after treatment. WOX5::GFP marks the quiescent center specific marker; J2341 marks columella stem cell. Images are representative of 12–16 plants in four replicate experiments treated as in Figure 1A. Bar = 50 µm. (B) Bright-field micrographs taken at five days after treatment. DR5::GUS marks response to endogenous auxin; Lugol staining marks amyloplasts; and CS31333 marks mature columella cells. Images are representative of 12–16 plants in four replicate experiments treated as in Figure 1A. Bar = 50 µm.
Figure 5.
Ammonium reduces the gravitropic response of primary roots.
(A) Primary root curvature in response to gravistimulation. Seedlings were cultured as described in Figure 1A. One day after transferred to 2-layer split plate medium, the seedlings were rotated 90° and root curvature was measured over time. Data represent means ± SD for 16 plants obtained in four replicate experiments. Means for nitrate and ammonium treatment are significantly different: ** p<0.01; *** p<0.001. (B) DR5::GUS expression pattern in root cap in response to gravistimulation. DR5::GUS plants were cultured as described in Figure 1A. Five days after transfer, the seedlings were rotated 90°. Plants were stained with GUS reaction buffer at the indicated times. Red arrow indicates the lower side of the root apex. Images are representative of 10–12 stained plants in three replicate experiments. Scale bars = 50 µm.
Figure 6.
Expression of AUX1 and PIN2 is down-regulated in response to ammonium treatment.
(A) AUX1::GUS. Bright-field images of root tips are representative of 10–12 plants in three replicate experiments, treated as for Figure 1A, and taken at the indicated days after transfer (DAT). Bar = 50 µm. (B) PIN2::PIN2-GFP. Confocal fluorescence images of PI-stained root tips, representative of 9–12 plants in three replicate experiments. Seedlings were treated as in Figure 1A, and tested at the indicated days after transfer (DAT). Bar = 50 µm. (C) and (D), Relative gene expression level of (C) AUX1 and (D) PIN2 was determined by quantitative real-time PCR. Plants were treated as in Figure 1A. The apical 5 mm segments of primary roots were sampled 8 h after transfer. At least 102 to 108 seedlings from three replicate experiments were collected for total RNA extraction. Bars plot means ± SD.
Figure 7.
The effect of ammonium on primary roots growth in aux1-7 and eir1-1 mutants.
(A) to (C), Primary root growth rates for (A) wild type, (B) aux1-7, and (C) eir1-1. Plants were treated as in Figure 1A. Data represent means ± SE (n = 4) from four replicate plates, with 4 seedlings each. (D) Relative primary root growth rates for the indicated genotypes. The data of root growth rates were re-plotted as a percent of plants treated with 1 mM nitrate respectively.