A Small-Molecule Screen Identifiesl-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth inArabidopsis   

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.

ETHYLENE-INSENSITIVE3 Is a Senescence-Associated Gene That Accelerates Age-Dependent Leaf Senescence by Directly Repressing miR164 Transcription in Arabidopsis

“…After 36 d, most of the estradiol-treated leaves became fully senesced or died, while untreated leaves were only partly yellowish ( Figure 4A). We also found that the transgenic plants constitutively overexpressing EIN3-GFP in the ein3 eil1 ebf1 ebf2 (qm) background (He et al, 2011) also displayed early senescence phenotypes compared with the qm mutant (see Supplemental Figure 4 …”

“…A transgenic reporter line that harbors the GUS gene driven by five tandem repeats of the EIN3 binding site (EBS) followed by a minimal 35S promoter (5XEBS:GUS/Col-0) has been used to monitor the transcriptional activity of EIN3 (Stepanova et al, 2007;He et al, 2011). It has been reported that senescence symptoms usually start from the tip and outer edge of a rosette leaf at a given age (Guo and Gan, 2006).…”

“…Thus, we propose a model to describe the signaling integration mediated by EIN3 (Figure 9), wherein EIN3 expression is induced by age as well as dark treatment, while its protein activity is enhanced by ethylene and JA, two common hormones in response to a wide array of environmental stresses (Dong, 1998). The abundance or activity of EIN3 protein is also regulated by other signals, such as auxin (He et al, 2011), gibberellin , light (Zhong et al, 2009), cold (Shi et al, 2012), and Glc , all of which are involved in the modulation of plant senescence (Sarwat et al, 2013). It will thus be interesting to investigate whether EIN3 plays a role in mediating and integrating these signals into the complex gene network that controls leaf senescence.…”

Biosynthesis of the Halogenated Auxin, 4-Chloroindole-3-Acetic Acid

“…In contrast, on the basis of liquid chromatography RTs relative to that of IAA, previous aminotransferase assays appear to have yielded mainly the enol form of IPyA . Tao et al (2008), Stepanova et al (2008), and He et al (2011) all report the production of IPyA from in vitro Trp aminotransferase assays, but not the phenomenon of tautomerization. However, an unidentified broad peak is visible in the UV chromatograms presented by Tao et al (2008) and He et al (2011), which might represent the keto tautomer of IPyA.…”

“…Tao et al (2008), Stepanova et al (2008), and He et al (2011) all report the production of IPyA from in vitro Trp aminotransferase assays, but not the phenomenon of tautomerization. However, an unidentified broad peak is visible in the UV chromatograms presented by Tao et al (2008) and He et al (2011), which might represent the keto tautomer of IPyA. Nevertheless, the in vitro assays were conducted under conditions that favor the keto form, as indicated by our NMR analyses.…”