Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis - PubMed (original) (raw)

Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis

Joshua I Armstrong et al. Proc Natl Acad Sci U S A. 2004.

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Abstract

Auxin modulates diverse plant developmental pathways through direct transcriptional regulation and cooperative signaling with other plant hormones. Genetic and biochemical approaches have clarified several aspects of the auxin-regulated networks; however, the mechanisms of perception and subsequent signaling events remain largely uncharacterized. To elucidate unidentified intermediates, we have developed a high-throughput screen for identifying small molecule inhibitors of auxin signaling in Arabidopsis. Analysis of 10,000 compounds revealed several potent lead structures that abrogate transcription of an auxin-inducible reporter gene. Three compounds were found to interfere with auxin-regulated proteolysis of an auxin/indole-3-acetic acid transcription factor, and two impart phenotypes indicative of an altered auxin response, including impaired root development. Microarray analysis was used to demonstrate the mechanistic similarities of the two most potent molecules. This strategy promises to yield powerful tools for the discovery of unidentified components of the auxin-signaling networks and the study of auxin's participation in various stages of plant development.

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Figures

Fig. 1.

Fig. 1.

A high-throughput screen for auxin signaling inhibitors. (A) BA3 seeds expressing GUS from an auxin-sensitive promoter were arrayed into 96-well microfilter plates (5-10 seeds per well) and grown in liquid culture for 5 days. Incubation of the seedlings with 5 μM NAA results in the tissue-specific expression of GUS in the root elongation zone, easily visualized after incubation with 5-bromo-4-chloro-3-indolyl β-

d

-glucuronide (X-gluc). The inclusion of an inhibitor of auxin signaling prevents GUS expression. (B) Structures of the four inhibitors, compounds A-D, chosen for detailed analysis.

Fig. 2.

Fig. 2.

The compounds inhibit auxin transcriptional activation in the BA3 and DR5::GUS reporter lines. Five-day-old BA3 (A and B) or DR5::GUS (C and D) seedlings were subjected to the indicated concentrations of IAA and compound for 4 h, followed by staining with X-gluc (A and C) or fluorimetric determination of GUS activity (B and D). Error = SE of the mean (n = 6).

Fig. 3.

Fig. 3.

The inhibitory activity is independent of auxin structure and reversible for most compounds. (A) Five-day-old BA3 seedlings were incubated with the indicated concentration of inhibitor and auxin for 4 h, followed by staining with X-gluc. 2,4-D, 2,4-dichlorophenoxyacetic acid. (B) Five-day-old BA3 seedlings were incubated with the indicated concentration of inhibitor (24-h wash) or inhibitor/IAA (prewash) for 4 h. The seedlings were then stained with X-gluc (prewash) or incubated in fresh medium before an additional 4-h treatment with IAA and subsequent staining with X-gluc (24-h wash).

Fig. 4.

Fig. 4.

Compounds A-C inhibit auxin-mediated proteolysis, and compounds A and B inhibit primary root elongation. (A) Seven-day-old HS::AXR3NT-GUS seedlings were heat shocked at 37°C for 2 h, followed by a 20-min incubation at 23°C. The indicated concentration of compound and/or NAA was added to the medium, and the seedlings were incubated for an additional 2 h (-NAA) or 40 min (+NAA) before quantification of GUS activity (Lower). Error = SE of the mean (n = 6). (Upper) Root tips from a representative seedling of each treatment stained with X-gluc. (B) Seedlings (Col-0) were germinated and grown vertically on medium containing increasing concentrations of compound A or B. (Upper) Primary root length was measured after 8 days of growth. Error = sample SD (n > 12). (Lower) Representative seedlings from each inhibitor treatment.

Fig. 5.

Fig. 5.

The transcriptional effects of inhibitor treatment. (A) Venn diagrams specify the number of 2-fold differentially regulated genes (P < 0.001) (induced or repressed) when comparing the indicated data sets. (B) Log2 expression values for selected auxin-induced genes from the microarray analysis for compounds A and B. Seven-day-old liquid culture-grown seedling clusters (Col-0) were treated for 1 h with IAA and/or inhibitor as indicated. (C) Semiquantitative RT-PCR analysis of the selected genes by using RNA from excised root tissue of BA3 seedlings treated with IAA and/or inhibitor for the indicated times. ACT8, ACTIN8.

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