Screening of small-molecule compounds that alter leaf-vein patterns and the auxin response in A. thaliana
In an attempt to identify small-molecule compounds that can affect leaf vein patterns, we screened a small-molecule compound library containing 3800 compounds, using 10-day-old seedlings of A. thaliana line Q0990. The expression of a vascular precursor cell reporter in Q0990 seedlings enabled us to screen for chemically induced defects in vascular development [17]. Seeds of Q0990 were grown in 96-well plates, in which each well contained a 10 μM solution of different compounds. Among these compounds, we selected 11 small molecules having an effect on vein pattern and leaf shape, all of which promoted central-vein thickening and an increase in the number of parallel vascular bundles (Fig. 1a, b).
To determine whether these physiological effects were correlated with a molecular auxin response, we employed transgenic Arabidopsis seedlings in which the synthetic auxin response element DR5 was fused to the green fluorescent protein (GFP) reporter. Among the 11 initially selected chemicals, we identified six candidates by confocal microscopy, in which the expression level of the reporter gene increased significantly after treatment, and the distribution of which was consistent with the location of leaf vein formation (Fig. 1c, d). Here, we focused on analyzing the character of the piperazine compound 1-[(4-bromophenoxy) acetyl]-4-[(4-fluorophenyl) sulfonyl] piperazine (ASP) and used it as a chemical tool to study the auxin signaling pathway.
Notably, we found that ASP showed structural similarities to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and endogenous IAA, both of which contain an unsaturated aromatic ring and a carboxylic acid side chain (Fig. 1e). This structural similarity reminded us of the functional similarity.
ASP can inhibit the elongation of primary and lateral roots, promote root hair development, and reduce gravitropism in A. thaliana
Phytohormones such as auxin and ethylene play crucial roles in the regulation of root growth [18]. To further characterize ASP activity, we accordingly performed a series of root assays. Following treatment of 6-day-old seedlings of wild-type A. thaliana (Col-0) with different concentrations of ASP and 2,4-D, we found that both compounds had clear concentration-dependent inhibitory effects on root growth (Fig. 2a, b) (Additional file 1: Fig. S1). When we measured primary root length at 24-h intervals, we found that the rate of primary root cumulative growth was reduced in the 5 μM ASP and 30 nM 2,4-D treatments (Fig. 2c). Furthermore, we observed that 30 nM 2,4-D and 4 μM ASP had similar effects on primary root growth (Fig. 2a, b) (Additional file 1: Fig. S1), and that under these treatments, the inhibition rate of primary root growth was 50%. Therefore, in subsequent experiments, 30 nM 2,4-D was used as positive control treatment.
Then we observed the lateral root growth in 9-day-old seedlings, we found that 10 nM 2,4-D increased the lateral root density, however, 30 nM 2,4-D inhibited such roots formation (Fig. 2d). When seedlings were treated with 2 μM and 5 μM ASP, we observed the emergence of numerous lateral roots on the primary root, although lateral root elongation was inhibited (Additional file 2: Fig. S2). Given that the effect of 10 nM 2,4-D and 2 μM ASP had similar effects on the lateral root density of WT, we speculated that although ASP promoted lateral root initiation, it inhibited lateral root elongation.
Gravitropism is the process whereby plants orientate their root growth toward gravity, and is a necessary response that ensures roots grow down through the soil [19]. We examined whether ASP had an effect on the gravitropic response of Col-0 seedlings by rotating seedlings grown vertically on 2 μM ASP-containing medium, and accordingly found that ASP retarded the rate of the gravitropic response. Thus, at 12 h after treatment, reorientation of the roots of ASP-treated plants showed noticeable inhibition, with a growth angle of 53° compared with the 70° of control plant roots. This trend became increasing more pronounced with continued root growth, and when final measurements were taken at 72 h after treatment, we found that the primary roots of ASP-treated plants had not bent to an angle of 90° (Fig. 2e, Additional file 3: Fig. S3).
Root hair development has been shown to be regulated by multiple plant hormones [20], and in the present study, we observed an increase in the number of root hairs growing on Col-0 seedlings in response to 1–5 μM ASP treatment; further, the length of these root hairs changed significantly (Fig. 2f, g). Moreover, we observed that the growth of root hairs occurred closer to the root tips (Additional file 4: Fig. S4). These findings thus indicated that ASP can inhibit primary and lateral root growth, retard the gravitropic response, and promote root hair production and growth in WT Arabidopsis seedlings.
Based on the effect of ASP on root growth, we suspected that root inhibition was due to the toxicity of ASP. We tested this possibility by using propidium iodide (PI)-stained root tip cells of 5-day-old seedlings treated with different concentrations of ASP, IAA and 2,4-D. PI is a nucleic-acid stain that can only penetrate cells with damaged or leaking cell membranes [21]. Confocal images revealed no detectable cellular damage due to treatment with ASP, IAA or 2,4-D, thereby indicating that the ASP-induced changes in root morphology are not associated with DNA damage or cell death (Additional file 5: Fig. S5).
ASP promotes hypocotyl elongation in A. thaliana
In general, well-known auxins (e.g., IAA, 2,4-D, 1-NAA) have no discernable effects on hypocotyl elongation [22]. In the present study, however, we found that treatment of A. thaliana with different ASP concentrations promoted hypocotyl elongation; furthermore, it had no clear concentration-dependent effects on 5-day-old seedlings (Fig. 3a, Additional file 1: Fig. S1). We observed semi-thin sections of hypocotyl tissue using light microscopy and measured cell length after 7 days of treatment with ASP to examine its effect on hypocotyl elongation at the cellular level. Longitudinal sections revealed that the hypocotyl cells of ASP-treated plants were significantly longer than those of control plants (Fig. 3b, Additional file 6: Fig. S6). Accordingly, these observations indicated that ASP enhanced hypocotyl growth by promoting cell elongation.
ASP has differing effects on auxin signaling mutants, and can induce the differential expression of several key genes in the auxin signaling pathway
To further explore whether ASP acted through the auxin signaling pathway, we assayed the phenotypes of three auxin signaling mutants, namely, tir1–1, axr2–1 and aux1–7. Treatment with 1–5 μM ASP increased the hypocotyl length in tir1–1 and aux1–7 relative to controls; and all three mutants showed no obvious root growth reduction. Furthermore, the promotion effects on the number and length of root hair were weakened in these mutants (Additional file 7: Fig. S7, Additional file 8: Fig. S8). In treatment with 4 μM ASP that inhibited root elongation rate by 50%, hypocotyl length of WT seedlings actually increased, with the promotion effect in tir1–1 and aux1–7 mutants being similar or larger than in the WT (Fig. 4a). However, compared with WT, the inhibitory effect in primary root growth was not significant in tir1–1, axr2–1 and aux1–7 (Fig. 4b). Meanwhile, the root hair number and length in tir1–1 and aux1–7 had no obvious changes by 4 μM ASP treatment, indeed in axr2–1 they showed significant reduction (Fig. 4c, d). From these results we concluded that the sensitivity of tir1–1 and aux1–7 to ASP in the root was different from that in the hypocotyl; therefore, we hypothesized that ASP affected root and hypocotyl growth via different pathways.
In addition, we used seedling lines containing the auxin response element DR5 fused to the β-glucuronidase (GUS) reporter to examine whether the effects of ASP were correlated with the auxin response. Histochemical staining of DR5::GUS seedlings grown for 24 h in the presence of 5 μM ASP revealed strong GUS expression in the cotyledons (Fig. 5a), the root/stem transition zone (Fig. 5b), and root tips (Fig. 5c). It is well known that auxin inhibits root growth and promotes root hair development [23]; thus, increased auxin accumulation implies root inhibition and root hair promotion.
On the basis of the aforementioned results, we hypothesized that ASP might induce auxin-like responses, and accordingly sought to examine the ability of ASP to induce auxin-response genes in 6-day-old seedlings. To this end, we performed real-time quantitative PCR (RT-qPCR) to analyze the expression of early auxin-response genes including, Aux/IAA, GH3 and SAUR within 2 h of treatment with ASP.
As expected, we found that ASP significantly up-regulated the auxin-responsive genes in WT seedlings. IAA2 expression increased at 15 min by IAA treatment and then decreased over the following 2 h. However, with ASP treatment, the expression level increased at 1 and 2 h sampling-time points. The trend of the relative expression of the GH3.5 was generally consistent under IAA and ASP treatment. The increase in gene expression level was evident after 1 h treatment, and an over four-fold increase was observed after 2 h of ASP treatment. The response of SAUR23 to IAA and ASP was rapid. After 15 min of treatment, the expression level had increased significantly (Fig. 6). Although the expression patterns of these genes were not exactly unanimous, the altered expression levels of the examined genes indicated that ASP treatment triggered auxin activity.
