Article Figures & Data
Figures
- Figure 1.
LR Development Is Affected by Expression of ABI4: abi4 Mutants Develop More LRs.
(A) Twelve-day-old seedlings of wild-type (WT) and the specified abi4 mutants grown on solid 0.5× MS medium. Bar = 5 mm.
(B) and (C) Roots of 4-d-old wild-type and abi4-1 mutant seedlings, respectively. Arrowhead indicates emerging LR. Bars = 100 μm.
(D) Average length of PRs in 12-d-old seedlings. The data represent the mean ± se of n = 3 independent experiments each containing 50 plants per treatment. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P < 0.05).
(E) Density of LRs at all developmental stages (including LRP) in 12-d-old seedlings. Statistical analysis performed as in (D).
[See online article for color version of this figure.]
- Figure 2.
ABI4 Expression in Roots.
(A) to (J) Expression patterns in roots.
(A) Two-week-old Arabidopsis seedlings expressing ABI4:GUS stained histologically for GUS activity.
(B) to (G) Enlarged views of different developmental stages of the PRs and LRs (from top to bottom regions).
(B) Mature PR near the shoot-root junction.
(C) Mature LR.
(D) Newly developed LR.
(E) Emerging LR.
(F) Root hair zone. Note the expression gradient within this zone.
(G) Root tip and meristematic zone.
(H) Section of GUS-stained mid-upper zone (8 mm from the shoot-root junction) of PR counterstained with ruthenium red.
(I) Cross section of PR in the upper zone (3 mm from the shoot-root junction), treated as in (H).
(J) Colored scheme indicating the different cell types in the section depicted in (I).
Bars = 1 mm in (A), 50 μm in (B) to (H), and 10 μm in (I) and (J).
- Figure 3.
ABI4 Expression Is Hormone Regulated.
(A) qRT-PCR analysis of ABI4 transcripts in 2-week-old wild-type plants treated for 4 h with 20 μM of the specified agents. The data represent the mean ± se of n = 3 independent experiments. Bars with different letters represent statistically different values using Tukey’s HSD post-hoc test (P ≤ 0.05).
(B) to (K) GUS staining of roots of 2-week-old ABI4:GUS/wild-type plants treated as in (A).
(B) to (F) Upper root zone.
(G) to (K) Root tips. Bars = 50 μm.
- Figure 4.
Root Structure of ABI4-Overexpressing Arabidopsis.
(A) Twelve-day-old seedlings of wild-type plants (WT) , 4C-S5 plants transformed with empty vector (Vec), or two lines of ABI4 overexpressors (OE2 and OE5) germinated and grown on 0.5× MS plates with (+) or without (−) 2 μM Dex. Bar = 5 mm.
(B) Density of PRs and LRs from the LRP stage of 12-d-old seedlings. The data represent the mean ± se of n = 3 independent experiments each containing 50 plants per treatment. White bars, −Dex; gray bars, +Dex. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P < 0.05).
[See online article for color version of this figure.]
- Figure 5.
Stage-Specific Inhibitory Effects of ABI4 on LR Development.
(A) and (B) LRs were counted in roots of 12-d-old seedlings in each of four developmental stages (De Smet et al., 2003): Stage A, initials with up to two layers of cells; stage B, three or more layers of cells just prior to emergence; stage C, immediately after emergence but <0.5 mm long; stage D, LRs longer than 0.5 mm.
(A) Effects of ABI4 overexpression on LR number: wild-type plants (black bars), 4C-S5 plants transformed with empty vector (white bars), or three lines of ABI4 overexpressors (gray bars) were germinated on 0.5× MS plates with (transgenic plants) or without (wild type) 2 μM Dex.
(B) Effects on LR number in abi4 mutants: the wild type, abi4-1, abi4-102, and abi4-103 mutants were germinated on 0.5× MS plates.
(C) Initial growth rates of LRs of abi4 mutants. The lengths of the first two visible LRs of 1-week-old vertically grown seedlings were measured. The added lengths at 24 and 48 h are presented. The data represent the mean ± se of n = 3 independent experiments each containing 20 plants per treatment. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P ≤ 0.05).
- Figure 6.
Interaction of ABI4 with Auxin.
(A) to (I) LR formation in control and IAA-treated wild-type (WT) and abi4 mutants. One-week-old seedlings were transferred to 0.5× MS medium ([A] to [D]) or to 0.5× MS supplemented with 20 μM IAA ([E] to [H]). Photographs were taken 1 week later. Bars = 0.5 mm.
(A) and (E) The wild type.
(B) and (F) abi4-1 mutant.
(C) and (G) abi4-102 mutant.
(D) and (H) abi4-103 mutant.
(I) Number of LRs was determined in the lower 5-mm section of the roots shown in (E) to (H).
(J) to (Q) One-week-old seedlings transformed with vector only ([J] to [M]) or pOp6:ABI4 ([N] to [Q]) were transferred to 0.5× MS containing, where indicated, 10 μM Dex and/or 20 μM IAA. Bottom sections of the roots were photographed 1 week later. Bars = 0.5 mm.
(J) and (N) Untreated.
(K) and (O) +Dex.
(L) and (P) +IAA.
(M) and (Q) +Dex and IAA.
(R) Number of LRs was determined in the lower 5-mm section of IAA treated roots. Gray bars, +IAA no Dex; black bars, +IAA +Dex. The data represent the mean ± se of n = 3 independent experiments each containing 50 plants per treatment.
- Figure 7.
ABI4 Affects Auxin and Cytokinin Distribution.
(A) to (C) and (E) to (P) GUS staining.
(A) to (C) Roots of seedlings of DR5:GUS/wild type.
(E) to (H) DR5:GUS/abi4-1 mutant.
(I) to (L) DR5:GUS/ABI4 overexpressor.
(M) and (N) ARR5:GUS/wild type.
(O) and (P) ARR5:GUS/abi4-1 mutant. Bars = 50 μm.
(D) Number of stained foci in DR5:GUS/wild-type and DR5:GUS/abi4-1 mutant seedlings. The data represent the mean ± se of n = 3 independent experiments each containing 50 plants per treatment.
- Figure 8.
abi4 Plants Are Insensitive to ABA and Cytokinin.
(A) and (B) One-week-old seedlings of the indicated lines were transferred to fresh 0.5× MS medium containing 10 μM ABA (A) or 10 μM zeatin (B). Plants were photographed 1 week later. 1, wild type; 2, abi4-1 mutant; 3, abi4-102 mutant; 4, abi4-103 mutant. Bars = 5 mm.
(C) LR density in abi4 mutants. White bars, no treatment control; gray bars, ABA treatment; black bars, zeatin treatment. The data represent the mean ± se of n = 3 independent experiments each containing 20 plants per treatment. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P ≤ 0.05). WT, wild type.
[See online article for color version of this figure.]
- Figure 9.
ABI4 Affects PIN1 Distribution.
(A) to (G) PIN1-GFP was visualized in PR tips of 12-d-old PIN1:PIN1-GFP/wild-type ([A] to [C]), PIN1:PIN1-GFP/ABI4 overexpressor (D), and PIN1:PIN1-GFP/abi4 mutant ([E] to [G]) seedlings with a fluorescent microscope.
(A), (D), and (E) Untreated seedlings.
(B) and (F) Treated with 10 μM ABA for 24 h.
(C) and (G) Treated with 10 μM zeatin for 24 h. Bars = 20 μm.
(H) to (J) PIN1-GFP expression in untreated emerging LRs.
(H) PIN1:PIN1-GFP/wild type.
(I) PIN1:PIN1-GFP/ABI4 overexpressor.
(J) PIN1:PIN1-GFP/abi4 mutant. Bars = 20 μm.
(K) and (L) Quantification of the fluorescence intensity.
(K) Images shown in (A), (D), and (E) were scanned transversally using ImageJ 1.42q software. Presented pattern was taken 60 μm above root tips. Similar patterns were obtained when other sections were analyzed.
(L) Pixel intensities of four root images were determined using ImageJ 1.42q. Ratios of the values obtained for ABA-treated (white bars) or zeatin-treated (gray bars) plants to that of nontreated plants were calculated for both wild-type and abi4-1 background. The data represent the mean ± se of n = 4. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P ≤ 0.05).
- Figure 10.
Polar Auxin Transport.
Agarose blocks containing 10 μM [3H]NAA were placed on the shoot-root junction of the indicated 7-d-old seedlings. When indicated, blocks contained additional 10 μM TIBA. Roots were analyzed 10 h later as described in Methods. The data represent the mean ± se of n = 3 independent experiments each containing 15 plants per treatment. WT, wild type.
- Figure 11.
A Possible Mechanism for Modulation of LR Development by ABI4.
A simplified model is shown for the hormonal regulation of LR development in Arabidopsis. ABI4 expression is enhanced by ABA and cytokinin and repressed by auxin. ABI4, in return, inhibits polar auxin transport in the root by decreasing the levels of the auxin efflux carrier PIN1, resulting in decreased auxin levels. Thus, ABI4 and auxin form a feedback-inhibition loop. In addition to ABI4-dependent pathway, ABA and cytokinin can act in an ABI4-independent way (dashed lines). Auxin is essential for LR formation and for the elongation of emerged LRs; thus, reduction in auxin levels results in inhibition of LR formation. Arrows and T-bars represent enhancement and inhibition, respectively.
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