The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

Abstract

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.

Figure 1.

Schematic Representation of the Auxin Biosynthesis Pathways in Arabidopsis. Classical view (A) and updated view (B) of the pathway modified to reflect the findings of this study. Solid arrows indicate single steps, whereas dashed arrows imply possible multiple steps. Arrows, gene names, and intermediates shown in gray versus black indicate hypothetical versus well-characterized steps, enzymatic activities, and metabolites, respectively. AMI, INDOLE-3-ACETAMIDE HYDROLASE; HTAM, N-hydroxyl TAM; IAAld, indole-3-acetaldehyde; IGs, indole glucosinolates; NIT, NITRILASE.

Figure 2.

The cyp79b2/b3 Double Mutant Can Suppress the High-Auxin Phenotypes of sur2, but Not That of TAA1:YUC1. Col, Col TAA1:YUC1, sur2, cyp79b2/b3, TAA1:YUC1 cyp79b2/b3, and sur2 cyp79b2/b3 were grown in plates for 3 d in the dark (A), 3 d in the dark followed by 4 d in constant light (B), or for 3 weeks in soil under a 16-h light/8-h dark cycle (C). Representative plants of each genotype are shown.

Figure 3.

Simultaneous Loss of Function of Multiple YUC and TAA1/TAR Family Members Results in Less-Than-Additive Phenotypic Defects in Quadruple, Quintuple, and Sextuple Knockout Mutants. Col, yuc1/2/4/6, wei8-1 tar2-1, wei8-1 tar2-1 yuc1/6, wei8-1 tar2-1 yuc1/2/6, wei8-1 tar2-1 yuc1/4/6, and wei8-1 tar2-1 yuc1/2/4/6 plants were grown for 3 d in the dark on AT plates supplemented with 10 μM ACC (A) or for 3 d in the dark on ACC-supplemented plates followed by 15 d in constant light on unsupplemented AT plates (B). The genotype of every plant shown was determined by PCR as described in Methods.

Figure 4.

Kyn, a Chemical Inhibitor of the TAA1/TAR Trp-Aminotransferase Activity, Blocks the High-Auxin Phenotypes of YUC1ox and TAA1:YUC1 Lines. Col, Col YUC1ox, Col TAA1:YUC1, and sur2 plants were grown in plates for 3 d in the dark (A) or for 3 d in the dark followed by 4 d in constant light (B) in the presence of 0, 1, 5, 10, 25, 50, or 100 μM Kyn. Representative plants are shown.

Figure 5.

Functional TAA1 and TARs Are Required for YUC1-Mediated Auxin Biosynthesis. Col, wei8-2, wei8-2 tar1-1, wei8-2 tar2-1, wei8-2 tar1-1 tar2-1, and yuc1/2/4/6 with or without the TAA1:YUC1 transgene were grown on plates for 3 d in the dark (A), on plates for 3 d in the dark followed by 4 d in constant light (B), or in soil for 4 weeks under a 16-h light/8-h dark cycle (C). Representative plants are shown.

Figure 6.

Quantification of the Levels of IAA and IAA Catabolites/Conjugates. Levels of free IAA (A), oxIAA (B), IAAsp (C), and IAGlu (D) were quantified in 2-week-old plants of Col, wei8-2, wei8-2 tar2-1, and yuc1/2/4/6 with and without the TAA1:YUC1 transgene. The bars represent average levels (± sd) of three independent biological replicates. The tables below the bar graphs indicate P-values of the genotype comparisons in an ANOVA analysis. *, **, and *** correspond to P-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively. F.W., fresh weight.