The piperazine compound ASP activates an auxin response in Arabidopsis thaliana

Abstract

Background: Auxins play key roles in the phytohormone network. Early auxin response genes in the AUX/IAA, SAUR, and GH3 families show functional redundancy, which makes it very difficult to study the functions of individual genes based on gene knockout analysis or transgenic technology. As an alternative, chemical genetics provides a powerful approach that can be used to address questions relating to plant hormones.

Results: By screening a small-molecule chemical library of compounds that can induce abnormal seedling and vein development, we identified and characterized a piperazine compound 1-[(4-bromophenoxy) acetyl]-4-[(4-fluorophenyl) sulfonyl] piperazine (ASP). The Arabidopsis DR5::GFP line was used to assess if the effects mentioned were correlated with the auxin response, and we accordingly verified that ASP altered the auxin-related pathway. Subsequently, we examined the regulatory roles of ASP in hypocotyl and root development, auxin distribution, and changes in gene expression. Following ASP treatment, we detected hypocotyl elongation concomitant with enhanced cell elongation. Furthermore, seedlings showed retarded primary root growth, reduced gravitropism and increased root hair development. These phenotypes were associated with an increased induction of DR5::GUS expression in the root/stem transition zone and root tips. Auxin-related mutants including tir1-1, aux1-7 and axr2-1 showed phenotypes with different root-development pattern from that of the wild type (Col-0), and were insensitive to ASP. Confocal images of propidium iodide (PI)-stained root tip cells showed no detectable damage by ASP. Furthermore, RT-qPCR analyses of two other genes, namely, Ethylene Response Factor (ERF115) and Mediator 18 (MED18), which are related to cell regeneration and damage, indicated that the ASP inhibitory effect on root growth was not attributable to toxicity. RT-qPCR analysis provided further evidence that ASP induced the expression of early auxin-response-related genes.

Conclusions: ASP altered the auxin response pathway and regulated Arabidopsis growth and development. These results provide a basis for dissecting specific molecular components involved in auxin-regulated developmental processes and offer new opportunities to discover novel molecular players involved in the auxin response.

Keywords: ASP; Auxin response; Auxin signaling; Chemical genetics; Phytohormone.

Fig. 1

Chemical genetics screening identified a novel compound ASP that produced abnormal leaf-vein pattern. a-b: Leaf vein pattern of 10-day-old seedlings of Q0990 line grown on 1/2 MS medium (a) and 1/2 MS medium with 10 μM ASP (b). c-d: Observation of 10-day-old seedlings of DR5::GFP reporter line grown on 1/2 MS medium (c) and 1/2 MS medium with 10 μM ASP (d) by confocal scanning laser microscope. E: Chemical structural formula of endogenous auxin indole-3-acetic acid (IAA), synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), and small-molecule compound 1-[(4-bromophenoxy) acetyl]-4-[(4-fluorophenyl) sulfonyl] piperazine (ASP)

Fig. 2

ASP decreased root growth in A. thaliana. a: Dose-response curve for wild type (Col-0) seedlings on ASP. Seedlings were treated for six days with different concentrations of ASP from 0 to 30 μM as indicated on X axis. b: Dose-response curve for Col-0 seedlings on 2,4-D. The concentrations of 2,4-D were from 10 nM to 10 μM as indicated on X axis. c: The cumulative rate of primary root length measured at 24-h interval. Seedlings were treated with 5 μM ASP and 30 nM 2,4-D respectively. DMSO was used as control. Each point represented the mean of at least 20 measurements. Error bars indicated the standard deviation. d: Effect of ASP and 2,4-D on lateral root density of WT seedlings. Seedlings were grown on 1/2 MS medium supplemented with 10 nM 2,4-D, 30 nM 2,4-D, 2 μM ASP and 5 μM ASP for nine days. DMSO was used as control (number of samples N = 10, P < 0.05). e: ASP reduced the gravitropic response. 2 μM ASP was used for gravitropism test. Col-0 seedlings grown on agar medium were rotated 90° at time 0. The angle of curvature from the horizontal was measured at the times indicated. Each point represents the mean of 24 measurements. Error bars indicate the standard deviation. f and g: Root hair of Col-0 seedlings response to ASP and 2,4-D. Seedlings vertically grown on the medium supplemented with 0–5 μM ASP and 30 nM 2,4-D for six days. Root hair upward root tips 5 mm was selected to measure and count (number of samples N = 50, P < 0.05). f: Statistics of root hair number. g: Measurements of root hair length

Fig. 3

ASP promoted hypocotyl elongation in Col-0 seedlings. a: Measurements of hypocotyl length in five-day-old seedlings under ASP treatment. The concentrations of ASP application were indicated on X axis. b: Statistical results of hypocotyl cell length. Hypocotyl cells were from semi-thin longitudinal section cut (N = 60, P < 0.05)

Fig. 4

Effects of ASP in Arabidopsis auxin signaling mutants. Wild-type and tir1–1, axr2–1 and aux1–7 mutants were grown on 1/2 MS medium supplemented with 4 μM ASP for six days. DMSO was used as control. a: Hypocotyl length. b: Primary root length. Data of A and B represent Means ± SD (N = 30, P < 0.05). c: Root hair number. d: Root hair length. Data of C and D represent Means ± SD (N = 3, P < 0.05). The experiment was repeated three times with similar results. * P < 0.05, ** P < 0.01, *** P < 0.001 or different letters indicate means statistically different at P < 0.05

Fig. 5

Effect of ASP on auxin response in Arabidopsis. Seedlings grown on 1/2 MS agar medium with DMSO for 5 days and then transferred into Eppendorf tube (10 seedlings per tube) containing 1 ml of 1/2 MS liquid medium supplied with DMSO, 1 μM indole-3-acetic acid (IAA) and 5 μM ASP and incubated for 24 h. After ten hours of β-glucuronidase (GUS) staining seedlings were cleared for microscopy analysis. GUS- expressing in cotyledon (a), root/ stem transition (b), and root tip (c) treated for 24 h with DMSO (Left), 1 μM IAA (Middle) and 5 μM ASP (Right). Photographs show representative individuals from at least 20 stained plants (Scale bar = 100 μm)

Fig. 6

RT-qPCR expression analyses of early auxin response genes. Six-day-old seedlings were treated with 5 μM ASP and 5 μM IAA for 0.5–2 h. Expression level shown are the Means ± SD from three biological replicates for each. Different letters indicate significant differences at P < 0.05