The power of chemical genomics to study the link between endomembrane system components and the gravitropic response

Abstract

Chemical genomics is a powerful approach to dissect processes that may be intractable using conventional genetics because of gene lethality or redundancy. Recently, a link has been established between endomembrane trafficking and gravitropism. To understand this link, we screened a library of 10,000 diverse chemicals for compounds that affected the gravitropism of Arabidopsis seedlings positively or negatively. Sixty-nine of 219 compounds from the primary screen were retested, and 34 of these were confirmed to inhibit or enhance gravitropism. Four of the 34 compounds were found to cause aberrant endomembrane morphologies. The chemicals affected gravitropism and vacuole morphology in concert in a tissue-specific manner, underscoring the link between endomembranes and gravitropism. One of the chemicals (5403629) was structurally similar to the synthetic auxin 2,4-dichlorophenoxy acetate, whereas the other three chemicals were unique in their structures. An in vivo functional assay using the reporter beta-glucuronidase under the auxin-inducible DR5 promoter confirmed that the unique compounds were not auxins. Interestingly, one of the unique chemicals (5850247) appeared to decrease the responsiveness to auxin in roots, whereas another (5271050) was similar to pyocyanin, a bacterial metabolite that has been suggested to target the endomembranes of yeast. These reagents will be valuable for dissecting endomembrane trafficking and gravitropism and for cognate target identification.

Fig. 1.

Screen for chemicals that affected gravitropism. The chemical library was screened in a 24-well format, and seedlings were scored for gravitropic response after reorientation. Chemicals dissolved in 20% DMSO were added to wells. Control wells contained an equivalent concentration of the solvent. The gravity vector is indicated by an arrow next to the g (on the right).

Fig. 3.

Several chemicals inhibit gravitropism in a tissue-specific manner. Dose-response experiments were done in the presence of 5403629 (A), the control auxin 2,4-D (B), 5271050 (C), 585247 (D), and 6220480 (E) at the concentrations indicated. Four-day-old seedlings grown in the presence of compounds were gravistimulated for 24 h, and the angle of curvature from a vertical position was measured. Solid lines indicate hypocotyl angles; dashed lines indicate root angles. All points are the mean ± standard error of at least duplicate experiments (n = 21-78 measurements per point). The hypocotyl and root-tip angles, respectively, of control seedlings without chemicals were: 68.0 ± 3.1, 88.3 ± 2.5 (A); 67.8 ± 3.1, 88.6 ± 2.0 (B); 68.8 ± 2.6, 79.7 ± 1.9 (C); 63.8 ± 2.3, 74.9 ± 2.9 (D); and 70.7 ± 3.6, 91.0 ± 3.9 (E). Chemical structures are shown in Table 1.

Fig. 2.

Four compounds affect vacuole morphology or targeting of the reporter molecule to the tonoplast in GFP:δ-TIP seedlings. Hypocotyls (A, C, E, G, and I) and roots (B, D, F, H, and J) were examined by laser-scanning confocal microscopy after germination and growth on the chemicals for 7 days in the light. Chemicals: (C and D) 5403629 at 6.6 μM, (E and F) 5271050 at 3.7 μM, (G and H) 5850247 at 35 μM, and (I and J) 6220480 at 30 μM final concentration. Chemical 5403629 was inhibitory to growth at the concentration tested, so seedlings were grown on medium without the chemical for 4 days then treated with the compound for 48 h. The arrows in C and D indicate vesiculations; E, aggregates; H, endoplasmic reticulum patterning; and J, aggregates. (A and B) Images of a control seedling grown in the presence of a concentration of DMSO equivalent to that of treated seedlings are shown for comparison. (Bars, 20 μm.)

Fig. 4.

Functional assay for auxin activity and auxin transport inhibition. Arabidopsis DR5::GUS seedlings were grown in the absence of chemicals; transferred to MS media containing 0.02% DMSO or chemicals 5403629 (3.3 μM), 5271050 (3.7 μM), 5850247 (35 μM), or 6220480 (30 μM); then stained for GUS activity 5 h later. (A) IAA and chemical 5403629 displayed intense staining, indicating DR5::GUS induction, whereas staining of 5271050-, 5850247-, and 6220480-treated seedlings were similar to the control (DMSO), indicating only basal induction of DR5::GUS. (B) Seedlings were pretreated with chemicals, 0.2% DMSO, or 100 μM TIBA for 14 h as in A; however, an agar block containing IAA was placed at the root tip (+IAA samples), and seedlings were stained for GUS activity 5 h later. (C) Test for auxin responsiveness. Seedlings were pretreated with chemicals and then incubated in a 1-μM IAA solution. (Bars, 0.2 mm.)