A small-molecule screen identifies L-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis

Abstract

The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Figure 1.

Low Concentrations of Kyn Suppress Root-Specific Phenotypes of ctr1-1 and eto1-2. (A) Three-day-old etiolated seedlings of Col-0, ctr1-1, and eto1-2 grown on MS medium or MS medium supplemented with 1 μM Kyn. (B) and (C) Quantification of the hypocotyl lengths (B) and root lengths (C) of seedlings shown in (A). Bars represent the average length (±sd) of at least 20 seedlings (Student’s t test, between Kyn-treated and nontreated seedlings; ***P < 0.001). [See online article for color version of this figure.]

Figure 2.

Kyn Inhibits EIN3 Nuclear Accumulation in Roots. (A) Quantification of the root lengths of 3-d-old etiolated Col-0, ctr1-1, eto1-2, and EIN3ox (an EIN3-overexpressing transgenic line) seedlings grown on MS medium supplemented with 10 μM ACC and/or 1.5 μM Kyn (K). The bars represent the average length (±sd) of at least 20 seedlings (Student’s t test, between ACC and ACC+Kyn treatment; ***P < 0.001). (B) GUS staining of EBS:GUS in the roots of Col-0, ctr1-1, and EIN3ox. The seedlings were grown on MS medium supplemented with 10 μM ACC and/or 1.5 μM Kyn. Bar = 100 μm. (C) GFP fluorescence of 35S:EIN3-GFP in the roots of ein3 eil1 double (dm) and ein3 eil1 ebf1 ebf2 quadruple (qm) mutants. The seedlings were grown on MS medium in the dark for 3 d and then transferred into liquid MS medium supplemented with 100 μM ACC and/or 100 μM Kyn and incubated in dark for 3 to 4 h.

Figure 3.

Kyn Decreases DR5:GUS Expression, but IAA Overrides Its Effect. (A) Three-day-old etiolated seedlings of Col-0, dm 35S:EIN3-GFP, and qm 35S:EIN3-GFP grown on MS medium with or without Kyn (1.5 μM) and/or ACC (10 μM). (B) Quantification of the root lengths of seedlings shown in (A) (Student’s t test, between ACC and ACC+Kyn treatments for dm EIN3-GFP and qm EIN3-GFP and between Kyn-treated and nontreated qm EIN3-GFP; ***P < 0.001). (C) Expression of the DR5:GUS reporter in the roots of Col-0, ctr1-1, ein2-5, and ein3 eil1 backgrounds. The seedlings were grown in the dark for 3 d on MS medium with or without Kyn (1.5 μM) and/or ACC (10 μM). Bar = 100 μm. (D) Expression of the DR5:GUS reporter in the roots of 3-d-old etiolated Col-0 seedlings treated with Kyn (100 μM) and/or IAA (10 μM) for 3 h. Bar = 100 μm. (E) Root lengths of Col-0, ctr1-1, eto1-2, and EIN3ox etiolated seedlings grown on MS medium in the presence of a range of IAA concentrations with or without Kyn (1.5 μM). Bars represent the average length (±sd) of at least 20 seedlings.

Figure 4.

Kyn Inhibits Ethylene-Induced IAA Biosynthesis. (A) IAA levels in the roots of 3-d-old etiolated seedlings grown on ACC (10 μM) and/or Kyn (1.5 μM) plates. Bars represent mean ± sd (n = 3, Student’s t test, between ACC and ACC+Kyn treatments; ***P < 0.001). FW, fresh weight. (B) GUS staining in the roots of ASA1:GUS and ein2-5 ASA1:GUS. Bar = 100 μm. (C) GFP fluorescence in the roots of TAA1:GFP-TAA1. Bar = 50 μm. (D) GUS staining in the roots of TAR2:GUS. Bar = 50 μm. The 3-d-old etiolated seedlings in (B) to (D) were grown on MS medium or MS supplemented with Kyn (1.5 μM) and/or ACC (10 μM). (E) Root lengths of Col-0, wei2-1, wei7-2, and wei8-1. The 3-d-old etiolated seedlings were grown on MS medium with different combinations of Kyn (1.5 μM), ACC (0.5 μM), and Trp (10 μM). Bars represent the average length (±sd) of at least 20 seedlings (Student’s t test, between ACC+Trp and ACC+Trp+Kyn treatments; ***P < 0.001).

Figure 5.

Kyn Potently and Competitively Inhibits TAA1/TAR1 Activity. (A) Kyn competitively inhibits TAA1 activity in vitro. The K_m and V_max for TAA1 using Trp as a substrate (top). K_i for the Kyn-mediated inhibition of TAA1 activity was determined using a Dixon plot (bottom). (B) The chemical structure of Kyn (top) and Trp (bottom). (C) Kyn is a substrate of TAA1. HPLC analysis of the Kyn and KYNA standards and of the reaction products when Kyn (500 μM) is used as a substrate with purified recombinant GST-TAA1 or boiled GST-TAA1. Absorbance at 280 nm is shown. (D) TAA1 loss-of-function mutants are hypersensitive to Kyn, while transgenic plants with multiple copies of TAA1 are hyposensitive to Kyn. Dose–response curves for the relative root lengths of three wei8 mutant alleles (top) and three independent transgenic TAA1:GFP-TAA1 lines (bottom). All seedlings were grown for 3 d in dark on MS medium supplemented with ACC (10 μM) and Kyn (from 0 to 1 μM), and the root lengths of respective genotypes grown on MS were used as controls. Bars represent the average length (±sd) of at least 30 seedlings. (E) Kyn also inhibits TAR1 activity and is a TAR1 substrate. HPLC analysis of Trp, IPyA, IAA, Kyn, and KYNA standards and of the reaction products catalyzed by purified recombinant GST-TAR1. Absorbance at 280 nm is shown. All the enzymatic activities were assayed by the HPLC analysis and quantification of the IPyA reaction products. Each data point represents at least two independent samples. These experiments were repeated at least twice with similar results.

Figure 6.

Phenotypic Analysis Indicates the Similarity between Kyn-Treated Wild-Type and Untreated wei8 tar2 Seedlings. (A) Six-day-old seedling phenotype of Col-0, wei8-1, and wei8 tar2 grown on vertical plates supplemented with increasing Kyn concentrations. More than 10 seedlings of every plate were observed, and two representative seedlings are shown. (B) Cotyledon phenotype of 8-d-old Col-0 and wei8 tar2 seedlings grown on MS medium supplemented without or with increasing Kyn concentrations. Note that Col-0 seedlings treated with 100 μM Kyn showed a distinct cotyledon upward curve phenotype also observed in wei8 tar2. More than 10 seedlings of every plate were observed. Bar = 2 mm.

Figure 7.

Molecular Docking Reveals That Kyn Competitively and Selectively Inhibits TAA1/TAR Activity. (A) Molecular structure illustrating the interaction between Kyn and KAT1. (B) Molecular modeling of the interaction between Kyn and TAA1, illustrating that Kyn could fit within the catalytic pocket of TAA1. (C) Molecular modeling of the interaction between Trp and TAA1. The top panels of (A) to (C) show the interaction between the small ligands (in yellow) and the catalytic pockets of the receptors (in gray). Bottom panels show the key residues that contribute to the binding with these proteins. (D) The structure alignment of TAA1 and KAT1 shows that they are very similar at binding sites for small ligands. TAA1 is shown as a blue ribbon with yellow side chains, while KAT1 is shown as a green ribbon with orange side chains. Bottom panel shows the details of key amino acids of TAA1 and KAT1 responsible for recognizing and binding to Kyn and/or Trp. (E) and (F) The multiple sequence alignment data show that the core amino acids in (D) (Lys-217, marked by red triangle; Gly-30, Tyr-194, Asn-168, and Arg-363, tagged with red arrowhead) are all conserved in the Arabidopsis TAA1/TAR family (E), whereas Lys-217 is not conserved in Tyr aminotransferases (F). The dark-colored boxes correspond to identical and partially conserved amino acids.

Figure 8.

IAA Stabilizes EIN3 Accumulation in the Nuclei of Root Tissues. (A) GFP fluorescence in the elongation zones (top) and root tips (bottom) of ein3 eil1 35S:EIN3-GFP, showing that IAA stabilizes EIN3 accumulation. (B) Kyn suppresses ACC-induced EIN3-GFP accumulation, whereas IAA reverses Kyn-mediated suppression in ein3 eil1 35S:EIN3-GFP. (C) IAA can, but ACC cannot, augment EIN3-GFP accumulation in the ein2 eil3 eil1 background. (D) IAA and/or Kyn have no effect on EIN3-GFP accumulation in the ein3 eil1 ebf1 ebf2 background. Three-day-old etiolated seedlings of indicated genotypes were treated with Kyn (100 μM), ACC (100 μM), and/or IAA (10 μM) for 3 h before fluorescence microscopy.

Figure 9.

A Proposed Model Illustrating the Interaction between Ethylene Signaling and Auxin Biosynthesis in the Regulation of Root Elongation. Ethylene acts to stabilize EIN3 through its canonical signaling pathway, and the accumulated EIN3 transcription factor activates the expression of several auxin biosynthetic genes, including WEI2, WEI7, TAA1, and TAR2. WEI2 and WEI7 are required for Trp production, while TAA1 and TAR2 (and probably other TARs as well) convert Trp into IPyA, which eventually leads to the synthesis of IAA and the inhibition of root elongation. In addition to promoting ethylene biosynthesis, IAA is also able to enhance EIN3 stability probably by repressing EBF1/2-mediated EIN3 degradation, forming a positive feedback loop between ethylene signaling and auxin biosynthesis. Kyn has been identified in this study as a small compound that competitively and selectively inhibits the family of TAA1/TARs. [See online article for color version of this figure.]