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, 106 (13), 5430-5

Biochemical Analyses of indole-3-acetaldoxime-dependent Auxin Biosynthesis in Arabidopsis

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Biochemical Analyses of indole-3-acetaldoxime-dependent Auxin Biosynthesis in Arabidopsis

Satoko Sugawara et al. Proc Natl Acad Sci U S A.

Abstract

Auxins are hormones that regulate many aspects of plant growth and development. The main plant auxin is indole-3-acetic acid (IAA), whose biosynthetic pathway is not fully understood. Indole-3-acetaldoxime (IAOx) has been proposed to be a key intermediate in the synthesis of IAA and several other indolic compounds. Genetic studies of IAA biosynthesis in Arabidopsis have suggested that 2 distinct pathways involving the CYP79B or YUCCA (YUC) genes may contribute to IAOx synthesis and that several pathways are also involved in the conversion of IAOx to IAA. Here we report the biochemical dissection of IAOx biosynthesis and metabolism in plants by analyzing IAA biosynthesis intermediates. We demonstrated that the majority of IAOx is produced by CYP79B genes in Arabidopsis because IAOx production was abolished in CYP79B-deficient mutants. IAOx was not detected from rice, maize, and tobacco, which do not have apparent CYP79B orthologues. IAOx levels were not significantly altered in the yuc1 yuc2 yuc4 yuc6 quadruple mutants, suggesting that the YUC gene family probably does not contribute to IAOx synthesis. We determined the pathway for conversion of IAOx to IAA by identifying 2 likely intermediates, indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN), in Arabidopsis. When (13)C(6)-labeled IAOx was fed to CYP79B-deficient mutants, (13)C(6) atoms were efficiently incorporated to IAM, IAN, and IAA. This biochemical evidence indicates that IAOx-dependent IAA biosynthesis, which involves IAM and IAN as intermediates, is not a common but a species-specific pathway in plants; thus IAA biosynthesis may differ among plant species.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Proposed IAA biosynthetic pathway in plants. (A) Previously proposed IAA biosynthetic pathway. The YUC and CYP79B pathways that lead to IAOx synthesis are illustrated by blue and red arrows, respectively. (B) The IAA biosynthetic pathway proposed in this study. Each number indicates a proposed pathway for biosynthesis of IAA from TRP. The bold arrows indicate novel pathways converting IAOx to IAA. Dotted square indicates the IAOx metabolic pathway in Arabidopsis. The IAOx-dependent IAA biosynthetic pathway is shown in the gray squares. Cloned genes that potentially encode enzymes for IAA, CL, and IG biosyntheses are shown in italics.
Fig. 2.
Fig. 2.
In vivo feeding of 13C6-IAOx to cyp79b2 cyp79b3 and WT seedlings. (A) Incorporation of 13C6 into metabolites in cyp79b2–2 cyp79b3–2 seedlings fed with 3 μM and 10 μM of 13C6-IAOx. (B) Incorporation of 13C6 into metabolites in WT seedlings fed with 10 μM of 13C6-IAOx. Ten-day-old seedlings were fed with 13C6-IAOx for 24 h at 21 °C. Data are means ± SD, n = 2.
Fig. 3.
Fig. 3.
Effect of IAM on the growth of cyp79b2 cyp79b3 at high temperatures. (A–C) Seven-day-old seedlings of WT, cyp79b2–2 cyp79b3–2, and 10 μM IAM-fed cyp79b2–2 cyp79b3–2. Seedlings were germinated and grown at 26 °C. (D) Fresh weight of WT and cyp79b2–2 cyp79b3–2 seedlings grown in the presence of 0, 1, 2, 5, and 10 μM of IAM for 7 days. Data are means ± SD, n = 16. (Scale bar, 2 cm.)
Fig. 4.
Fig. 4.
Effect of IAM and IAN on the growth of Arabidopsis seedlings. (A–E) Two-week-old seedlings of WT, sur1–1, WT treated with 60 μM IAM, WT treated with 30 μM IAN, and WT treated with 60 μM IAM and 30 μM IAN. Arrows indicate outgrowth of numerous adventitious roots. (Scale bar, 3 mm.)

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