Article Figures & Data
Figures
- Figure 1.
Genomic Structure of T-DNA Insertion Alleles.
(A) Structure of the MDR4 gene. Boxes represent exons, and lines represent introns. The positions of the T-DNA insertions in mdr4-1 (SALK_010207) and mdr4-2 (SALK_072038) are represented by triangles.
(B) RT-PCR analysis of MDR4 transcript levels in Col-0 and the two independent mdr4 T-DNA alleles used in this study.
(C) Gene diagram of mdr1 T-DNA insertion alleles used in this study. Ws is the genetic background of the previously described mdr1-1 and mdr1-2 alleles. To obtain an allele in the Col-0 background, mdr1-3 (Salk_033455) was isolated.
(D) PCR results showing interruption of MDR1 and MDR4 in the mdr1-3 mdr4-1 double mutant (i to iv) and proper function of the gene-specific primers on wild-type DNA (v and vi). (i) MDR1 5′ primer plus MDR1 3′ primer gave no product. (ii) MDR1 5′ primer plus T-DNA Lb1a primer gave a product with the expected size. (iii) MDR4 5′ primer plus MDR4 3′ primer gave no product. (iv) MDR4 3′ primer plus T-DNA Lb1a primer gave a product with the expected size. (v) Wild-type DNA: product of the MDR1 5′ primer plus the MDR1 3′ primer was the expected size. (vi) Wild-type DNA: product of the MDR4 5′ primer plus the MDR4 3′ primer was the expected size.
- Figure 2.
Contribution of MDR1 and MDR4 to Acropetal and Basipetal Auxin Transport in Roots.
(A) Acropetal auxin transport measured by applying 3H-IAA to the root-shoot junction zone and later determining the amount of radioactivity in an apical portion of the root. Values shown are mean ± se of five independent trials, each involving eight roots per genotype. BA, benzoic acid, a molecule not acted on by the polar transport stream that was used as a control.
(B) Auxin transport assayed by induction of ProDR5:GUS. Acropetal: auxin applied at the junction zone activated GUS expression and quantified by MUG assay in more apical regions of the root. Baseline GUS activity was 860 ± 150 relative fluorescence units h−1 in the wild type and 590 ± 95 relative fluorescence units h−1 in mdr1. Basipetal: auxin applied at the root tip induced GUS expression in more basal portions of the root. Values are mean fold induction over mock treatment ± se, and n = 6 trials of 10 roots per genotype.
(C) Dose–response curve for ProDR5:GUS induction shows that roots of mdr1 and the wild type were similarly sensitive to auxin; mean ± se, n = 6 trials for each point, with 10 roots per measurement.
(D) Basipetal auxin transport measured by applying 3H-IAA to the root apex and later determining the amount of radioactivity in a basal segment of the root. Values shown are mean ± se of seven independent trials, each involving eight roots per genotype.
- Figure 3.
Sporadic Curvature and Altered Auxin Distribution in mdr1 Roots.
(A) Image series showing sporadic root curvature in two alleles of mdr1. Wild-type and mdr4 alleles display relatively straight root growth.
(B) Summation of absolute curvature in wild-type and mdr1 roots quantifies the phenotype displayed in (A). The absolute value of curvature at each point along the midline of the root was summed to create a measure of deviation from straight growth. On average, mdr1 roots display approximately threefold more curvature when growing vertically than the wild type. Plotted are mean values ± se of eight trials.
(C) Auxin distribution indicated by ProDR5:GFP and measured by laser scanning confocal microscopy in vertically grown wild-type and mdr1-3 roots. Four representative mdr1-3 examples show more GFP signal in the epidermis and portions of the root cap (arrowheads) than the wild type. Bar = 50 μm.
- Figure 4.
Gravitropism of Wild-Type, mdr1, and mdr4 Roots Quantified with Computational Morphometrics.
(A) Total angle of the root is plotted versus time after reorientation for the wild type (n = 6) and two alleles of mdr1 (n = 7). There is no effect of the mutation.
(B) Total angle of the root is plotted versus time after reorientation for the wild type (n = 8) and two alleles of mdr4 (n = 10) ± se. The mutants respond faster and to a greater extent than the wild type.
(C) Spatiotemporal distribution of gravitropic curvature (K) in the Ws wild type. Length of the root axis is plotted in the y-dimension, and time is plotted along the x-dimension. K is color-coded and plotted in the z-dimension. Straight areas of the root are shown in cool colors, and curvature is shown as warm colors, as shown by the horizontal color scale bar. Shown is the average of six [0] individual roots.
(D) Spatiotemporal distribution of gravitropic curvature of Col wild-type roots, with an average of eight individuals.
(E) and (G) Spatiotemporal distribution of gravitropic curvature of mdr1 roots, with an average of seven individuals. The mdr1 mutations did not affect the response.
(F) and (H) Spatiotemporal distribution of gravitropic curvature of mdr4 roots, with an average of 10 individuals. The area of main curvature is shifted basally relative to the wild type. The black contour lines demark areas where the difference between the mutant and the wild type is significant to a level of P = 0.05.
- Figure 5.
Gravity-Induced Auxin Asymmetry in Wild-Type and mdr4 Roots.
(A) ProDR5:GFP signal in wild-type and mdr4 roots before and after 8 h of reorientation. Images were obtained with a horizontally mounted epifluorescence microscope. Three example experiments are shown. The background auxin-dependent GFP signal in the elongation zone was generally higher in mdr4 roots, and the accumulation on the lower flank after gravitropism was more diffuse than in the wild type.
(B) Optical slices (longitudinal medial) through wild-type or mdr4 root apices expressing ProDR5:GFP obtained by laser scanning confocal microscopy after 6 h of gravitropism. A generalized root outline in red is superimposed for orientation because the intense ProDR5:GFP signal landmark at the root tip is omitted. Arrowheads at the left edge of each image point to the ProDR5:GFP signal along the lower flank of the root, which differs between the mutant and the wild type. Shown are three individuals that are representative of the six examined for each genotype.
(C) Cross sections computationally constructed from the z-series of confocal images from which the images in (B) were selected show greater ProDR5:GFP signal in the lower part of mdr4 roots compared with the wild type.
- Figure 6.
Characterization of mdr1 mdr4 Double Mutant Phenotypes.
(A) Gross phenotype of mdr1-3 mdr4-1 double mutants showing the epinastic cotyledons and wavy roots of the mdr1 single mutant.
(B) Summation of absolute curvature ± se to quantify the wavy root phenotype. The mdr1 (n = 7) and mdr1 mdr4 double mutants (n = 8) were not different in this respect.
- Figure 7.
Characterization of mdr1 mdr4 Gravitropism Using Computational Morphometrics.
(A) Total angle of mdr1 mdr4 (n = 6) roots accruing over time following reorientation ± se displayed the same hypertropic pattern as mdr4 single mutants, and like mdr1-1 and mdr1-2, mdr1-3 (n = 9) was indistinguishable from the wild type.
(B) Spatiotemporal distribution of gravitropic curvature of wild-type (Col) roots, with an average result of 16 individuals.
(C) Spatiotemporal distribution of gravitropic curvature of mdr1-3 roots, with an average of nine individuals.
(D) Spatiotemporal distribution of gravitropic curvature of mdr1-3 mdr4-1 roots, with an average of six individuals. The results demonstrate that mdr1-3 is indistinguishable from the wild type and that the double mutant behaves like mdr4. The black contour line shows where and when curvature is different from the wild type at a significance level of P = 0.05. In all phenotypes, no synergistic effects of the mutations were observed.
- Figure 8.
Gravitropism of tt4 Mutants and tt4 mdr4 Double Mutants.
(A) Total angle of wild-type, tt4, and tt4 mdr4 roots accruing over time following reorientation. The hypertropism of mdr4 prevailed over the hypotropism of tt4 in the tt4 mdr4 double mutant, indicating that mdr4 is epistatic to tt4, consistent with flavonoids being endogenous regulators of MDR4-dependent auxin transport. Shown are the mean values [MSOffice2] of 16 individuals for the wild type, six for tt4, and six for tt4 mdr4. Angle was determined from electronic images acquired every 2 min. The se indicated by error bars every 15 min.
(B) Spatiotemporal distribution of gravitropic curvature of Col-0 wild-type roots.
(C) Spatiotemporal distribution of gravitropic curvature of tt4 roots. Gravitropic curvature is weaker and shifted apically compared with the wild type in tt4 mutants.
(D) Spatiotemporal distribution of gravitropic curvature of tt4 mdr4 roots. The basal shift in the spatial distribution of curvature due to the mdr4 mutation is shown to be epistatic to the opposite effect of the tt4 mutation, as was the effect on total angle in (A). The black contour line surrounds the region that differs from the tt4 single mutant response to a statistically significant degree (P = 0.05).
- Figure 9.
Diagram of MDR-Dependent Auxin Streams in the Apex of the Primary Root.
MDR1 is expressed in the central cylinder and the cortex of the root apex, where auxin is transported acropetally and centripetally. These processes help balance expansion rates of cells on sides of the growing zone to guide vertical root growth. MDR4 is an important contributor to basipetal auxin transport. It plays a role in controlling differential growth during gravitropism, perhaps by affecting the auxin asymmetry that drives the process.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Movie 1 - Auxin asymmetry in gravistimulated wild-type root.
- Supplemental Movie 2 - Auxin asymmetry in gravistimulated mdr4 root.
