Small-Molecules Selectively Modulate Iron-Deficiency Signaling Networks in Arabidopsis

Abstract

Plant growth requires optimal levels of iron (Fe). Fe is used for energy production, numerous enzymatic processes, and is indispensable for cellular metabolism. Recent studies have established the mechanism involved in Fe uptake and transport. However, our knowledge of Fe sensing and signaling is limited. Dissecting Fe signaling may be useful for crop improvement by Fe fortification. Here, we report two small-molecules, R3 and R6 [where R denotes repressor of IRON-REGULATED TRANSPORTER 1 (IRT1)], identified through a chemical screening, whose use blocked activation of the Fe-deficiency response in Arabidopsis thaliana. Physiological analysis of plants treated with R3 and R6 showed that these small molecules drastically attenuated the plant response to Fe starvation. Small-molecule treatment caused severe chlorosis and strongly reduced chlorophyll levels in plants. Fe content in shoots was decreased considerably by small-molecule treatments especially in Fe deficiency. Small-molecule treatments attenuated the Fe-deficiency-induced expression of the Fe uptake gene IRT1. Analysis of FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) and subgroup Ib basic helix-loop-helix (bHLH) gene (bHLH38/39/100/101) expression showed that R3 affects the FIT-network, whereas R6 affects both the FIT and Ib bHLH networks. An assessment of the effects of the structural analogs of R3 and R6 on the induction of Fe-dependent chlorosis revealed the functional motif of the investigated chemicals. Our findings suggest that small-molecules selectively modulate the distinct signaling routes that operate in response to Fe-deficiency. R3 and R6 likely interrupt the activity of key upstream signaling regulators whose activities are required for the activation of the Fe-starvation transcriptional cascade in Arabidopsis roots.

Keywords: Arabidopsis thaliana; chemical biology; iron deficiency signaling; iron homeostasis; small-molecules.

FIGURE 1

Small-molecules block Fe-deficiency response. (A) Small-molecules inhibit Pro_IRT1:LUC expression. 6-days ½MS-grown plants were treated for 3 days under +Fe or -Fe media in the presence or absence of 50 μM indicated small-molecules. (B) IRT1 protein accumulation in response to small-molecules. The 10-days ½MS-grown plants were treated for 3 days under +Fe or -Fe in the presence or absence of small-molecules. IRT1 protein was detected in total protein extract of roots using an anti-IRT1 antibody. Samples were separated in the same gel. CB, coomassie blue stain. (C) The 2D structure of small-molecules. R3, N-[4-(1,3-benzothiazol-2-yl)-2-methylphenyl] acetamide; R6, 2-benzoyl-1-benzofuran-5-carboxylic acid.

FIGURE 2

Small-molecules cause severe Fe-deficiency chlorosis. (A) Phenotype of plants with small-molecule treatments. 9-days ½MS-grown plants were transferred to Fe50 or Fe0 media for 9 days in the presence or absence of 50 μM indicated small-molecules. (B) Total chlorophyll content of plants under small-molecules treatment. Plants were treated as in (A). Data are mean ± SD (n = 5). ^∗, significant change vs. mock at P < 0.05 by Student’s t test. FW, fresh weight.

FIGURE 3

Small-molecules affect Fe levels in plants. Effect of small molecules on Fe (A), Mn (B), and Zn (C) contents in shoots. 10-days ½MS-grown plants were treated with or without 50 μM indicated small-molecules under Fe50 or Fe0 condition for 10 days. Levels of elements were measured by ICP-OES. Data are mean ±SD (n = 3). Significant differences compared with mock by Student’s t test: ^∗, P < 0.05. DW, dry weight.

FIGURE 4

The expression of Fe-acquisition genes is inhibited by small-molecules. qPCR analysis of expression of IRT1 (A), FRO2 (B), and FIT (C) in roots. 9-days ½MS-grown plants were transferred to +Fe or -Fe in the presence or absence of 25 μM indicated small-molecules for 3 days. The expression of UBC21 was used to normalize mRNA levels. The gene expression levels in mock +Fe were set to 1. Data are mean ± SE (n = 3). Significant differences compared with mock by Student’s t test: ^∗, P < 0.05.

FIGURE 5

The expression of Ib bHLH genes is deregulated by R6 but not by R3. qPCR analysis of expression of bHLH38 (A), bHLH39 (B), bHLH100 (C), and bHLH101 (D) in roots. 9-days ½MS-grown plants were treated for 3 days under +Fe or -Fe in the presence or absence of 25 μM indicated small-molecules. The expression of UBC21 was used to normalize mRNA levels. The gene expression levels in mock +Fe were set to 1. Data are mean ± SE (n = 3). Significant differences vs. mock by Student’s t test: ^∗, P < 0.05.

FIGURE 6

GSNO or ACC or NAA did not rescue the inhibitory effect of R3 or R6 on Fe-deficiency response. (A) Reverting small-molecule effect by GSNO, ACC, and NAA. 8-days ½MS grown plants were transferred to -Fe medium with 25 μM small-molecules in the presence or absence of 100 μM GSNO or 10 μM ACC or 0.2 μM NAA for 2-days. (B) NO level is not affected by R3 or R6 or R7. 8-days ½MS grown plants were transferred to -Fe with or without 50 μM R3 or R6 or R7 for 4-days. The NO was imaged in root tip using 5 μM DAF-FM DA dye.

FIGURE 7

Effect of R3 structural derivatives. (A) The structural derivatives do not mimic the R3 effect. 9-days ½MS-grown plants were transferred to Fe50 or Fe0 medium for 9 days in the presence or absence of 50 μM small-molecules. (B) The 2D structure of R3 structural derivatives.

FIGURE 8

Effect of R6 structural derivatives. (A) The phenotype of plants under structural derivatives treatment. 9-days ½MS-grown plants were treated in Fe50 or Fe0 for 9 days in the presence or absence of 50 μM small-molecules. (B) The 2D structure of R6 structural derivatives. (C) IRT1 protein accumulation in roots under R6SD1 treatment. 10-days ½MS-grown plants were transferred to +Fe or -Fe with or without R6SD1 for 3 days. IRT1 protein was detected using an anti-IRT1 antibody. CB, coomassie blue stain.

FIGURE 9

Proposed model for the possible action of R3 and R6 in Fe-deficiency transcriptional-network of Arabidopsis. Fe-status determines the BTS/BTSL stability/function. Under Fe deficiency, BTS is likely degraded, thereby allowing the accumulation of IVc bHLH proteins, which activates the transcription of Ib bHLH genes (directly) and FIT (indirectly). R6 inhibits the Fe-deficiency-stimulated expression of these IVc bHLH targets. R6 may block the IVc bHLH function by directly affecting the stability or indirectly promoting the degradation through BTS action. In addition to these signals, an unknown Fe-deficiency born signal directly activates the FIT transcription. FIT forms heterodimer with each member of Ib bHLH transcription factors and regulate the Fe-uptake gene IRT1. R3 most likely intercepts this unknown signal, thus repressing the FIT expression. Solid lines indicate data-supported information from previous studies. Open arrow indicates translation. Proteins are represented by colored-ovals and the genes are indicated by colored-boxes.