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

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Abstract

Key message

The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

Abstract

The 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.

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Acknowledgments

We thank Ms. Emi Ishida for technical assistance and Dr. Shozo Fujioka for helpful discussion. A part of this work was supported by The Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (to Y. S. and K. S.), and a Grant-in-Aid for Scientific Research (nos. 23580144 and 26506015 to M. S. and no. 25514004 to C. Y.) from the Japan Society for the Promotion of Science. This paper is contribution no. 1015 from the Kihara Institute for Biological Research, Yokohama City University.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, 244-0813, Japan

    Masashi Suzuki, Chiaki Yamazaki, Marie Mitsui, Yusuke Kakei, Yuka Mitani, Ayako Nakamura & Yukihisa Shimada

  2. RIKEN Plant Science Center, Yokohama, Kanagawa, 230-0045, Japan

    Masashi Suzuki, Yuka Mitani & Yukihisa Shimada

  3. NARO/WARC, Zentsuji, Kagawa, 765-8508, Japan

    Takahiro Ishii & Kazuo Soeno

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  1. Masashi Suzuki
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  2. Chiaki Yamazaki
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Correspondence to Yukihisa Shimada.

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299_2015_1791_MOESM1_ESM.ppt

Supplementary material 1 (PPT 139 kb) Supplementary Fig. S1. The influence of NAA and 2,4-D on expression of the auxin responsive genes Aux/IAA1 and Aux/IAA19. (a) Scheme of growth conditions and chemical treatments. (b) The relative expression of these genes was analyzed using qRT-PCR in 7-day-old Arabidopsis seedlings treated with synthetic auxins for 3 h. White, gray, and black bars represent the control, 10 μM NAA, and 10 μM 2,4-D treatments, respectively. The transcript levels are presented as values relative to those of the control, defined as 1, after normalization to GAPDH levels. Data represent the mean ± se (n = 3). Statistically significant differences relative to the control are indicated by asterisks (Student’s t test; *P < 0.05)

299_2015_1791_MOESM2_ESM.ppt

Supplementary material 2 (PPT 143 kb) Supplementary Fig. S2. The influence of the auxin-biosynthetic inhibitor kynurenine on expression of the auxin-responsive genes Aux/IAA1 and Aux/IAA19. (a) Scheme of growth conditions and chemical treatments. (b) Relative expression of these genes was analyzed using qRT-PCR in 7-day-old Arabidopsis seedlings treated with 30 μM kynurenine and/or 3 μM IAA for 3 h. White and gray bars represent Aux/IAA1 and Aux/IAA19, respectively. The transcript levels are presented on a log2 scale as values relative to those of the control, defined as 0, after normalization to GAPDH levels. Data represent the mean ± se (n = 3). Statistically significant differences relative to the control are indicated by asterisks (Student’s t-test; *P < 0.05)

299_2015_1791_MOESM3_ESM.ppt

Supplementary material 3 (PPT 144 kb) Supplementary Fig. S3. Gene expression analysis of auxin responsive genes in the in vivo conversion experiment by IPyA feeding. (a) Scheme of growth conditions and chemical treatments. (b) Relative expression of the auxin-responsive genes Aux/IAA1 (white bar) and Aux/IAA19 (gray bar) was analyzed using qRT-PCR in 7-day-old Arabidopsis seedlings. The transcript levels are presented on a log2 scale as values relative to those of the control, defined as 0, after normalization to GAPDH levels. Data represent the mean ± se (n = 3). Statistically significant differences relative to the control are indicated by asterisks (Student’s t-test; *P < 0.05)

299_2015_1791_MOESM4_ESM.ppt

Supplementary material 4 (PPT 1292 kb) Supplementary Fig. S4. Characteristic phenotypes of auxin-deficient and -excess mutants. (a) Morphological phenotypes of 4-day-old seedlings of the wild-type (WT), wei8-1, wei8-1 tar2-1, YUC1ox (left), and 7-day-old seedlings of sur1-3 (right). Bars indicate 1 cm. (b) Relative expression of the auxin responsive genes Aux/IAA1 (white bar) and Aux/IAA19 (gray bar) was analyzed using qRT-PCR in 4-day-old seedlings of the WT, wei8-1, wei8-1 tar2-1, YUC1ox, and 7-day-old seedlings of sur1-3. The transcript levels are presented on a log2 scale as values relative to those of the WT, defined as 0, after normalization to GAPDH levels. Data represent the mean ± se (n = 4). Statistically significant differences relative to the WT are indicated by asterisks (Student’s t-test; *P < 0.05)

299_2015_1791_MOESM5_ESM.ppt

Supplementary material 5 (PPT 153 kb) Supplementary Fig. S5. Characterization of YUC1ox. The expression levels of YUC1 (a) and endogenous IAA levels (b) in YUC1ox grown for 5 days were compared with the wild type (WT). YUC1 expression was analyzed with qRT-PCR, and the endogenous IAA level was analyzed using LC–MS/MS. The transcript levels are presented as values relative to those of the WT, defined as 1, after normalization to GAPDH levels. Data represent the mean ± se (n = 3). Statistically significant differences relative to the WT are indicated by asterisks (Student’s t-test; *P < 0.05, # P < 0.1)

299_2015_1791_MOESM6_ESM.ppt

Supplementary material 6 (PPT 379 kb) Supplementary Table S1. Primers and TaqMan probes used for qRT-PCR or for cDNA amplification to generate YUC1ox

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Suzuki, M., Yamazaki, C., Mitsui, M. et al. Transcriptional feedback regulation of YUCCA genes in response to auxin levels in Arabidopsis . Plant Cell Rep 34, 1343–1352 (2015). https://doi.org/10.1007/s00299-015-1791-z

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