Allelic analyses of the Arabidopsis YUC1 locus reveal residues and domains essential for the functions of YUC family of flavin monooxygenases

Abstract

Flavin monooxygenases (FMOs) play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yuc1 yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yuc1 alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide (FAD) cofactor and the other binds to nicotinamide adenine dinucleotide phosphate (NADPH). We also identified the Ser(139) to Phe conversion in yuc1, a mutation that is located between the two nucleotide-binding sites. By analyzing a series of yuc1 mutants, we identified key residues and motifs essential for the functions of YUC1 FMO.

Figure 1. Cotyledon vascular patterns of yuc4 enhancers

(A) Wild type seedlings displayed four-loop vein pattern. (B and C) Seedlings of yuc1 yuc4 double mutants (T-DNA insertion lines). Note that the yuc1 yuc4 double mutants made fewer veins. Some of the veins were not continuous. Vascular patterns were variable in the yuc1 yuc4 double mutants, but they never formed lobes observed in wt cotyledons. (D to H) Seedlings of yuc4 enhancers. The enhancers were new yuc1 alleles (see text for details). Seedlings of yuc1–4 yuc4 (D), yuc1–5 yuc4 (E), yuc1–6 yuc4 (F), yuc1–7 yuc4 (G), and yuc1–8 yuc4 (H) are shown. Seedlings were grown for 7 days under long day conditions. Seedlings were cleared using acetic acid/ethanol treatments followed by chloral hydrate treatments.

Figure 2. Floral defects of yuc4 enhancers

(A) Adult plants of yuc4 enhancers, from left to right, wild type, yuc1 yuc4 (T-DNA insertion line), yuc1–4 yuc4, yuc1–5 yuc4, yuc1–6 yuc4, yuc1–7 yuc4, and yuc1–8 yuc4. (B) Inflorescence apex of the yuc4 enhancers. Note that all of the new yuc1 alleles produced abnormal flowers in the yuc4 background.

Figure 3. Conserved motifs in flavin monooxygenases (FMOs) and in YUC proteins

(A) The conserved motifs in all of FMOs. The putative FAD and NADPH binding sites are indicated. (B) Conserved motifs among Arabidopsis YUC proteins. In addition to the well conserved glycine residues in the FAD binding and NADPH binding sites, residues near the GXGXXG motifs appear to be conserved among YUC proteins. (C) Mutations that disrupt YUC1 function in Arabidopsis were indicated. Note that mutations in the conserved glycine residues disrupted YUC1 functions.