Article Figures & Data
Figures
- Figure 1.
Promoter–GUS Constructs and GUS Activity in Transgenic Arabidopsis Plants.
(A) Schematic representation of the AuxRD–GUS fusions. The transcription start site (+1) is shown, and the lengths of the promoter elements are indicated by the numbers over the constructs. CORE, nonauxin responsive segment spanning positions −92 to +96 of PS-IAA4/5 gene; UBQ, UBQ3 promoter.
(B) The mean GUS activity observed in seedlings analyzed without auxin treatment. The degree of auxin inducibility mediated by each promoter–GUS construct is shown as the ratio of GUS activity in seedlings treated with 2 × 10−5 M IAA relative to GUS activity observed in untreated seedlings. Each point is representative of seedlings from a single line.
(C) Typical GUS staining patterns seen in No-0 plants harboring the indicated promoter–GUS fusions. The specific lines shown contain −318–GUS (line 37), CORE–GUS (line 53), A–GUS (line 5.1), B–GUS (line 20), BA–GUS (line 1.3), 4×A–GUS (line 30.4), and 4×B–GUS (line 13.2). The seedlings were treated without (−IAA) or with (+IAA) 2 × 10−5 M IAA for 6 hr followed by staining with X-gluc.
- Figure 2.
Details of Histochemical Localization of GUS or GFP Reporter Gene Expression.
(A) Dark-field image of GUS staining in a 5-day-old WT/BA3 root after treatment with 10 −7 M IAA for 6 hr. The GUS staining reflects the expression of GUS in the elongation zone of the root tip.
(B) Cross-section through the apical region of a WT/BA3 root treated with 10−7 M IAA for 6 hr and then stained for GUS activity. The GUS staining pattern shows that the BA promoter mediates high levels of expression in the atricoblast cell file.
(C) Fluorescent image of the root tip from a 5-day-old wild-type seedling harboring the BA–GFP transgene treated with 2 × 10−5 M IAA for 24 hr.
(D) Bright-field image of a 5-day-old axr2/BA3 root seedling stained for GUS activity without being treated with IAA.
(E) Bright-field image of a 5-day-old axr2/BA3 seedling stained for GUS activity after being treated with 10−7 M IAA for 6 hr. Note the lack of staining in the elongation zone that is seen in WT/BA3 plants.
(F) Bright-field image of 5-day-old hls3/BA3 seedlings stained for GUS activity after being treated with 10−7 M IAA for 6 hr.
(G) Bright-field image of a 2-week-old hls3/BA3 seedling stained for GUS activity after being treated with 10−7 M IAA for 6 hr. Note the GUS staining in the emerging primordia of newly forming lateral roots.
- Figure 3.
Histochemical Analysis of GUS Activity in the Root Tip of BA–GUS Plants after Treatment with 0 to 10−4 M IAA.
Homozygous seedlings containing the BA–GUS gene in wild-type (WT), axr1-12 (axr1), axr2-1 (axr2), axr3-1 (axr3), axr4-2 (axr4), or aux1-7 (aux1) backgrounds were examined.
- Figure 4.
Outline of the Mutant Screen.
(A) Steps in the mutant screen. EMS, ethyl methanesulfonate.
(B) Two-week-old seedlings grown vertically in a four-celled Falcon 1009 Petri plate.
(C) Two-week-old seedlings with the agar removed from cell 2 so that the roots can be treated with auxin incubation solution.
(D) Simulation of the screening. A WT/BA3 seedling treated with 2 × 10−5 M IAA for 6 hr and stained for GUS activity was placed among the roots of seedlings incubated without IAA.
- Figure 5.
Characterization of age1 and age2.
(A) Schematic representation of the map location of the age1 and age2 loci. The genetic distances, in centimorgans, to the loci indicated were determined by analyzing 190 lines for the pvv4 marker, 195 lines for the ATEAT1 marker, 114 lines for the PAI1 marker, 43 lines for the ASA1 marker, and 88 lines each for the nga106 and nga139 markers. Chr., chromosome.
(B) to (E) Five-day-old wild-type (WT) and age1 seedlings ([B] and [C], respectively) grown on 1.6% agar plates inclined at 45°. (D) shows 4-week-old wild-type (left) and age1 (right) plants. (E) shows mature plants: wild type (left), age1 (center), and age2 (right). A comparison of (B) and (C) shows the decreased number of bends in the roots of wild-type versus age1 plants. (D) shows the decreased size and pale nature of age1 plants relative to wild-type plants. (E) shows the morphological phenotype of mature wild-type, age1, and age2 plants.
- Figure 6.
Histochemical Staining of WT/BA3, age1/BA3, and age2/BA3 Plants.
(A) GUS staining patterns in the roots of 5-day-old seedlings after a 6-hr treatment with either 10−8 or 10−7 M IAA. WT, wild type.
(B) GUS staining patterns in the aerial portion of the seedlings that had not been treated with IAA.
- Figure 7.
RNA Gel Blot Analysis of Wild Type, age1, or age2 Plants That Were Homozygous for Both BA–GUS and BA8–GFP.
(A) Total RNA was isolated from 5-day-old wild-type (WT) or age1 seedlings that had been treated with 0 to 10−4 M IAA. Twenty micrograms of RNA was separated by electrophoresis, blotted to a Nytran membrane, and probed with radiolabeled gene-specific probes for the indicated cDNAs.
(B) Twenty micrograms of RNA isolated from the roots of 5-day-old untreated age2 seedlings was analyzed as described for (A).
Tables
- Table 1.
Effect of Various Growth Regulators on GUS Expression in the Root Elongation Zone of the WT/BA3 Line
Treatment GUS Activitya Control − 20 μM IAA +++ 20 μMIBA + 20 μM NAA +++ 20 μM2,4-D +++ 20 μM 2,3-D − 20 μM PCIB − 20 μM TIBA − 20 μMFC − 20 μM indole − 20 μMtryptophan − 20 μM GA − 20 μM ABA − 20 μM BA − 20 μM SA − 20 μM ACC − 20 μL/L C2H4 − Air − -
↵a Batches of five to 10 seedlings were analyzed in at least two independent experiments. GUS activity in the root elongation zone of 5-day-old light-grown seedlings was assessed visually as strong (+ + +), weak (+), or undetectable (−).
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