Kazuo Soeno

Transcriptional feedback regulation of YUCCA genes in response to auxin levels in Arabidopsis.

Key messageThe IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.AbstractThe phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

Arabidopsis Seedlings Over-Accumulated Indole-3-acetic Acid in Response to Aminooxyacetic Acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.

Metabolic Conversion of Castasterone and Brassinolide into Their Glucosides in Higher Plants

Castasterone (CS) and brassinolide (BL) were administered to mung bean (Vigna radiata) explants, Arabidopsis thaliana seedlings, and cultured Catharanthus roseus cells, and the glucosylated metabolites were analyzed using LC/MS/MS. In mung bean and C. roseus, CS-2-O-glucoside (CS-2G), -3-O-glucoside (CS-3G), -22-O-glucoside (CS-22G), and -23-O-glucoside (CS-23G) were identified as metabolites of CS, whereas BL-2G, BL-3G, and BL-23G were identified as metabolites of BL. In A. thaliana, CS and BL were converted into their respective 2-O- and 23-O-glucosides. Of the metabolites identified with BL and CS administration, BL-23G was the predominant metabolite in mung bean and A. thaliana, whereas the 3-O-glucoside of BL was abundant in C. roseus. This is the first report of the metabolic conversion of CS into CS-2G, CS-3G, CS-22G, and CS-23G, and of BL into BL-2G and BL-3G. Our results indicate that the glucosylation profiles of BL and CS vary with plant species, and that the glucosylation of CS is rather limited quantitatively, compared with that of BL.

Biochemical and chemical biology study of rice OsTAR1 revealed that tryptophan aminotransferase is involved in auxin biosynthesis; identification of a potent OsTAR1 inhibitor, pyruvamine2031.

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

Suppression of Elongation and Growth of Tomato Seedlings by Auxin Biosynthesis Inhibitors and Modeling of the Growth and Environmental Response

To develop a growth inhibitor, the effects of auxin inhibitors were investigated. Application of 30 μM L-α-aminooxy-β-phenylpropionic acid (AOPP) or (S)-methyl 2-((1,3-dioxoisoindolin-2-yl)oxy)-3-phenylpropanoate (KOK1101), decreased the endogenous IAA levels in tomato seedlings at 8 days after sowing. Then, 10–1200 μM AOPP or KOK1101 were sprayed on the leaves and stem of 2–3 leaf stage tomato plants grown under a range of environmental conditions. We predicted plant growth and environmental response using a model based on the observed suppression of leaf enlargement. Spraying AOPP or KOK1101 decreased stem length and leaf area. Concentration-dependent inhibitions and dose response curves were observed. Although the effects of the inhibitors on dry weight varied according to the environmental conditions, the net assimilation rate was not influenced by the inhibitors. Accordingly, the observed decrease in dry weight caused by the inhibitors may result from decreased leaf area. Validation of the model based on observed data independent of the dataset showed good correlations between the observed and predicted values of dry weight and leaf area index.

Analysis of a putative auxin biosynthesis inhibitor, indole-3-oxoethylphosphonic acid, in Arabidopsis.

Previously we identified indole-3-acetic acid (IAA) biosynthesis inhibitors that act on the conversion of l-tryptophan to indole-3-pyruvic acid in the IAA biosynthesis of Arabidopsis. In the present study, we synthesized a new compound, indole-3-oxoethylphosphonic acid (IOEP), and found that IOEP had an inhibitory effect on IAA biosynthesis in Arabidopsis. The results suggest that IOEP is a novel inhibitor of auxin biosynthesis in Arabidopsis.

Auxin signaling through SCFTIR1/AFBs mediates feedback regulation of IAA biosynthesis

We previously reported that exogenous application of auxin to Arabidopsis seedlings resulted in downregulation of indole-3-acetic acid (IAA) biosynthesis genes in a feedback manner. In this study, we investigated the involvement of the SCFTIR1/AFB-mediated signaling pathway in feedback regulation of the indole-3-pyruvic acid-mediated auxin biosynthesis pathway in Arabidopsis. Application of PEO-IAA, an inhibitor of the IAA signal transduction pathway, to wild-type seedlings resulted in increased endogenous IAA levels in roots. Endogenous IAA levels in the auxin-signaling mutants axr2-1, axr3-3, and tir1-1afb1-1afb2-1afb3-1 also increased. Furthermore, YUCCA (YUC) gene expression was repressed in response to auxin treatment, and expression of YUC7 and YUC8 increased in response to PEO-IAA treatment. YUC genes were also induced in auxin-signaling mutants but repressed in TIR1-overexpression lines. These observations suggest that the endogenous IAA levels are regulated by auxin biosynthesis in a feedback manner, and the Aux/IAA and SCFTIR1/AFB-mediated auxin-signaling pathway regulates the expression of YUC genes.