The main auxin biosynthesis pathway in Arabidopsis

Abstract

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.

Fig. 1.

Proposed IAA biosynthesis pathway in plants. (A) Previously proposed IAA biosynthesis pathway. (B) The IAA biosynthesis pathway proposed in the present study. The bold arrows indicate proposed functions of TAA1 and YUC, respectively. The IAOx pathway is illustrated in a dotted square. IAA-Asp and IAA-Glu are IAA metabolites investigated in this study.

Fig. 2.

Phenotypes of TAA1 and YUC overexpression plants in Arabidopsis. (A) Ten-day-old seedlings of pER8, TAA1ox, yuc1D, and TAA1ox yuc1D and (B–E) magnification of stem–root junctions (Est treatment for 5 d). (F) Est-treated 10-d-old seedlings of pER8, TAA1ox, YUC6ox, and TAA1ox YUC6ox and (G–J) magnification of root tip region (Est treatment for 5 d). (Scale bars: 1 cm.)

Fig. 3.

Phenotypes of TAA-deficient and YUC-deficient mutants. (A) Three-week-old WT seedlings. (B) Upper region and (C) inflorescence of 7-wk-old WT plants. (D) Three-week-old seedlings of wei8-1 tar2-1 mutants. (E) Upper region and (F) inflorescence of 7-wk-old wei8-1 tar2-2 mutants. (G) Three-week-old seedlings of yuc1 yuc2 yuc4 yuc6 mutants. (H) Upper region and (I) inflorescence of yuc1 yuc2 yuc6 mutants. (Scale bars: 1 cm.)

Fig. 4.

The level of IPA in WT plants and TAA-deficient and YUC-deficient mutants. (A) Aerial parts of 3-wk-old seedlings grown in soil were used for IPA analysis. Values are mean ± SD (n = 4). (B) The buds of 7-wk-old plants before flowering were used for IPA analysis. Values are mean ± SD (n = 3). Differences between WT and mutants are statistically significant at P < 0.05 (*P < 0.05 and **P < 0.01, t test).

Fig. 5.

Conversion of IPA to IAA by YUC2. (A) The HPLC profile for authentic IPA with UV detection (328 nm). (B) The HPLC profile for authentic IAA, (C) GST-YUC2 reaction mixture, and (D) GST reaction mixture with fluorescence detection (280 nm excitation and 355 nm emission).