Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis

Abstract

Auxin biosynthesis in plants has remained obscure although auxin has been known for decades as a key regulator for plant growth and development. Here we define the YUC gene family and show unequivocally that four of the 11 predicted YUC flavin monooxygenases (YUC1, YUC2, YUC4, and YUC6) play essential roles in auxin biosynthesis and plant development. The YUC genes are mainly expressed in meristems, young primordia, vascular tissues, and reproductive organs. Overexpression of each YUC gene leads to auxin overproduction, whereas disruption of a single YUC gene causes no obvious developmental defects. However, yuc1yuc4, yuc2yuc6, all of the triple and quadruple mutants of the four YUC genes, display severe defects in floral patterning, vascular formation, and other developmental processes. Furthermore, inactivation of the YUC genes leads to dramatically reduced expression of the auxin reporter DR5-GUS in tissues where the YUC genes are expressed. Moreover, the developmental defects of yuc1yuc4 and yuc1yuc2yuc6 are rescued by tissue-specific expression of the bacterial auxin biosynthesis gene iaaM, but not by exogenous auxin, demonstrating that spatially and temporally regulated auxin biosynthesis by the YUC genes is essential for the formation of floral organs and vascular tissues.

Figure 1.

Overexpression of the YUC genes and analysis of the expression patterns of the YUC genes. (A) A phylogenetic tree of the YUC family of flavin monooxygenases. Each YUC is assigned a YUC number largely based on the order of their discoveries. The petunia YUC1 homolog FZY is also included. (B) Overexpression of the YUC genes leads to auxin overproduction. (Top panel from left to right) Wild type, iaaM, YUC1. (Bottom panel from left to right) YUC2, YUC4, and YUC6. (OX) Overexpression. (C) YUC1 expression. (C-1,C-2) YUC1 expression in the inflorescence apex and young flowers. (C-3,C-4) GUS staining of the YUC1∷GUS reporter lines. (D) YUC4 expression. (D-1) In situ hybridization of YUC4 in the inflorescence apex. (D-2,D-3,D-4) GUS staining of the YUC4∷GUS reporter lines. (E) YUC2 expression. (E-1) In situ hybridization of YUC2 in the inflorescence apex. (E-2–E-4) GUS staining of YUC2∷GUS reporter lines. (F) YUC6 expression. (F-1) In situ hybridization of YUC6 in the inflorescence apex. (F-2–F-4) GUS staining of the YUC6∷GUS reporter lines. Arrowheads indicate the center of the shoot meristem proper.

Figure 2.

Analysis of the loss-of-function yuc mutants. (A) Identification of T-DNA insertion mutants for the YUC genes. The T-DNA insertion sites were schematically indicated and the exact insertion sites shown as the distance from the ATG codon in the genomic sequence: yuc1, 1027 base pairs (bp); yuc 2, 1348 bp; yuc4-1, 245 bp; yuc4-2, 174 bp; and yuc 6, 1022 bp. (B) Mature plants. (Top, left to right) Wild type, yuc1yuc4, and yuc2yuc6. Both yuc4-1 and yuc4-2 in various combinations with other yuc mutants showed the same phenotypes. The phenotypic characterizations shown in this paper are based on the yuc4-1 allele. (Bottom, left to right) yuc1yuc2yuc4, yuc1yuc4yuc6, and yuc1yuc2yuc4yuc6. (C) Defects in plant stature and apical dominance. (From left to right) Wild type, yuc2yuc6, yuc1yuc2yuc6, yuc1yuc4, yuc1yuc2yuc4, yuc1yuc4yuc6, and yuc1yuc2yuc4yuc6.

Figure 3.

The roles of the YUC genes in floral organ patterning. (A) A wild-type flower with some sepals and petals removed. (B) A flower of yuc2yuc6 with some sepals and petals removed. The arrow points to the short stamens. (C–F) Flower-like structures of yuc1yuc4. The inflorescence apex of yuc1yuc4 is shown in C, and typical yuc1yuc4 flowers are displayed in D–F. The arrow in D indicates the ovule-like structure, the arrow in E points to the meristem-like protrusion, and the arrow in F refers to a petal-like organ. (G–I) Flowers of yuc1yuc2yuc4. The inflorescence apex is shown in G and typical flowers are shown in H and I. In I, a gynoecium-like structure grew out of the top of another gynoecium. (J–L) Various floral structures of yuc1yuc4yuc6. (M–O) Inflorescence apex of yuc1yuc2yuc4yuc6. Note the cylinder structure in M.

Figure 4.

Venation patterns in yuc flowers and leaves. (A–F) Flower venation patterns. (G–L) Vascular patterns in leaves. (A,G) Wild type. (B,H) yuc2yuc6. (C,I) yuc1yuc4. (D,J) yuc1yuc2yuc4. (E,K) yuc1yuc4yuc6. (F,L) yuc1yuc2yuc4yuc6.

Figure 5.

Effects of yuc mutations on the expression of DR5-GUS and complementation of yuc mutants with the iaaM gene. (A) DR5-GUS in wild-type background. (B) DR5-GUS in yuc1yuc2yuc6 background. (C) DR5-GUS in yuc1yuc4yuc6 background. (A–C) The top shows the whole seedling, and the bottom shows the true leaves. Arrowheads point to root tips. (D) A flower of yuc1yuc2yuc6 (left) and a flower of yuc1yuc2yuc6 transformed with the iaaM gene under the control of the YUC6 promoter (right). Note the differences in stamens (arrows). (E) The infertile phenotypes of yuc1yuc2yuc6 (top) were rescued by the expression of the iaaM gene (bottom). (F) The decreased apical dominance phenotypes of yuc1yuc2yuc6 (left) were complemented by the iaaM gene. (G) Complementation of the floral defects of yuc1yuc4 (left) by expressing the iaaM gene under the control of the YUC1 promoter (right). (H) The sterile phenotypes of yuc1yuc4 (left) were rescued by the iaaM gene (right).