Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis
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基因进展 2006年第13期
Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, California 92093-0116, USA
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.
[Keywords: Auxin; auxin biosynthesis; flavin monooxygenase; flower development; vascular development]
Received January 30, 2006; revised version accepted April 26, 2006.
Auxin is a key regulator for many aspects of plant growth and development including floral organ formation and vascular tissue patterning, but the underlying mechanisms are not well understood. It is generally believed that cell fate and organ formation in plants are determined by local auxin gradients, which are generated by PIN family proteins that control the directions of auxin flow and overall auxin distribution (Friml et al. 2002, 2003; Benkova et al. 2003; Reinhardt et al. 2003; Heisler et al. 2005; Leyser 2005). A key missing link in our understanding of auxin-regulated developmental processes is that the molecular mechanisms of auxin biosynthesis are not known. Without knowing where auxin is synthesized in plants, it is difficult to understand the exact mechanisms of auxin movements and how auxin gradients are created and maintained.
Although multiple pathways including several tryptophan-dependent routes and a tryptophan-independent pathway are proposed to synthesize auxin in plants, the molecular details and the physiological roles of each pathway are not known (Bartel 1997; Cohen et al. 2003). The only auxin biosynthesis pathway that is well defined both biochemically and genetically is the iaaM/iaaH pathway that was found in plant pathogens such as Pseudomonas and Agrobacterium, but not in plants (Kosuge et al. 1966; Comai and Kosuge 1982). The iaaM gene encodes a tryptophan-2-monooxygenase that catalyzes the conversion of tryptophan to indole-3-acetamide, which is then hydrolyzed to release indole-3-acetic acid (IAA) by the hydrolase iaaH (Kosuge et al. 1966). Genetic dissection of auxin biosynthesis in plants has proven very difficult, and no auxin-deficient mutant has ever been identified. However, there are several auxin overproduction Arabidopsis mutants including yucca (Zhao et al. 2001), sur1 (Boerjan et al. 1995; King et al. 1995), sur2 (Delarue et al. 1998), and CYP79B2 overexpression lines (Zhao et al. 2002). Both SUR1 and SUR2 are not directly involved in auxin biosynthesis, and mutations in the two genes lead to the diversion of indole-3-acetaldoxime from glucosinolate synthesis to auxin biosynthesis (Barlier et al. 2000; Mikkelsen et al. 2004). Indole-3-acetaldoxime is synthesized from tryptophan by the cytochrome P450 CYP79B2/B3, which appear not to play a critical role in plant development (Zhao et al. 2002).
We previously proposed that a flavin monooxygenase, encoded by the YUCCA1 (YUC1) gene, plays a key role in auxin biosynthesis through analysis of a dominant gain-of-function yucca mutant in Arabidopsis (renamed as yuc1D here) (Zhao et al. 2001). However, in the absence of loss-of-function mutants, it has been difficult to prove the role of YUC1 in Arabidopsis. Here we define the YUC gene family in Arabidopsis, examine the expression patterns of several YUC family members, and use reverse genetics to unequivocally show that these YUC flavin monooxygenases are involved in auxin biosynthesis. We show that disruption of certain combinations of four of the 11 predicted YUC genes (YUC1, YUC2, YUC4, YUC6) (Fig. 1A) in Arabidopsis causes dramatic developmental defects that are rescued by the bacterial auxin biosynthesis gene iaaM under the control of a corresponding YUC promoter, but not by applications of exogenous auxin. Furthermore, we show that the YUC genes are not ubiquitously expressed and inactivation of the YUC genes leads to decreased expression of an auxin reporter DR5-GUS in the cells where the YUC genes are expressed. These data provide unambiguous evidence that the loss-of-function yuc phenotypes are caused by local auxin deficiency and that the YUC flavin monooxygenases are key auxin biosynthesis enzymes. More importantly, we provide evidence demonstrating that spatially and temporally regulated auxin biosynthesis is a key determinant essential for the formation of floral organs and vascular tissues, and other developmental processes. Moreover, the temporal and spatial patterns of auxin biosynthesis now can be inferred from the expression patterns of the YUC genes, providing a necessary foundation for analysis of auxin dynamics in plants.
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.
Results
Overexpression of YUC genes leads to auxin overproduction
The Arabidopsis genome contains 10 YUC1-like genes (Fig. 1A). In this study, we focus our analysis on the roles of YUC1 and the three closest YUC1 homologs (YUC2, YUC4, and YUC6) in auxin biosynthesis and plant development (Fig. 1A). To test whether these four YUC genes function in auxin biosynthesis, we constructed transgenic Arabidopsis plants that overexpress the cDNA of each of these YUC genes under the control of the Cauliflower Mosaic Virus 35S promoter. As shown in Figure 1B, overexpression of each YUC gene led to phenotypes indistinguishable from those of yuc1D and the iaaM overexpression lines, suggesting that overexpression of each YUC gene also caused auxin overproduction. Therefore, it is likely that the YUC-like genes in this study also play a role in auxin biosynthesis because of their sequence homology with YUC1 and the observed auxin overproduction phenotypes in the overexpression lines.
Expression patterns of YUC1, YUC2, YUC4, and YUC6 in the inflorescence apex and flowers
To uncover the roles for each YUC gene in plant development, we analyzed the expression patterns of these four YUC genes by RNA in situ hybridization and by histochemistry of transgenic lines harboring promoter–GUS (-Glucuronidase) fusions. YUC4, the gene with highest sequence identity to YUC1 in Arabidopsis (Fig. 1A), shows expression patterns similar to that of YUC1 in the inflorescence apex (Fig. 1C-1, D-1). Both YUC1 and YUC4 are expressed in the apical meristems and young floral primordia (Fig. 1C-1, D-1). Initiation of flower primordia correlates well with the accumulation of YUC1 and YUC4 mRNAs (Fig. 1C-1, D-1). YUC1 is also expressed in the floral meristems and at the basal end of the young floral buds (Fig. 1C-1, C-2). It is also evident that YUC4 is expressed in the floral meristems (Fig. 1D-1). Consistent with the in situ hybridization data, the YUC1::GUS lines showed strong staining in the floral meristems and at the base of the floral organs (Fig. 1C-3). At later stages of flower development, the GUS staining of the YUC1::GUS lines is localized in discrete groups of cells in both stamens and carpels (data not shown). In the fully matured flowers, the expression of the GUS reporter in YUC1::GUS lines is completely shut down (Fig. 1C-4). The YUC4::GUS lines also show GUS staining patterns similar to those observed from the in situ hybridization (Fig. 1D-1, D-2). Significant GUS staining was observed at the base of the floral organs in young flower buds (Fig. 1D-2). In late stages of flowers, the GUS staining of YUC4::GUS lines was found predominantly in the apical regions of the carpels, stamens, and sepals (Fig. 1D-3). In mature flowers, the GUS staining was limited only to parts of the gynoecium (Fig. 1D-4). Unlike YUC1 and YUC4, YUC2 and YUC6 show very low expression in the inflorescence apex (Fig. 1E,F). However, weak staining was observed throughout the inflorescence apex including the stems of the YUC2::GUS lines (Fig. 1E-2). The YUC2::GUS lines also show staining in the young flower buds, petals, stamens, and gynoecium of young flowers (Fig. 1E-3). Staining in late stages of flowers in YUC2::GUS lines is mainly restricted to the valves of the gynoecium (Fig. 1E-3, E-4). Unlike the YUC2::GUS lines, the YUC6::GUS lines have staining mainly in stamens and pollen (Fig. 1F). The YUC genes are not ubiquitously expressed; instead, their expression is rather temporally and spatially restricted, suggesting that normal auxin functions may be dependent on the locations of auxin production.
Expression of the YUC genes in other tissues
The public Arabidopsis microarray databases (https://www.genevestigator.ethz.ch) indicate that the four YUC genes are also expressed in other tissues (Zimmermann et al. 2004) (Supplementary Fig. 1). Our promoter::GUS lines also showed that the YUC genes have distinct staining patterns in seedlings and young leaves (Supplementary Fig. 2). Strong GUS staining was observed at the base of young leaf primordia in YUC1::GUS lines, whereas GUS staining was observed in discrete spots of both basal and apical regions of young leaves in YUC4::GUS lines (Supplementary Fig. 2). We did not observe GUS staining in mature leaves in YUC1::GUS lines, but weak staining was observed in higher-order vascular strands in YUC4::GUS plants (Supplementary Fig. 2). GUS staining in YUC2::GUS lines was evident in young leaf primordia and adult leaves (Supplementary Fig. 2). No GUS staining was observed in seedlings of YUC6::GUS lines.
Identification of loss-of-function mutants for the YUC1, YUC2, YUC4, and YUC6 genes
We identified T-DNA insertion mutants for the four YUC genes from the T-DNA mutant collections (Fig. 2A; Tissier et al. 1999; Alonso et al. 2003) and carried out phenotypic analysis of the identified T-DNA mutants, but none of the mutants displayed any obvious developmental defects (data not shown). These observations are consistent with our hypothesis that there are overlapping functions among the YUC family members (Zhao et al. 2001). We did not observe any PCR products when we used primers flanking the T-DNA insertion sites to perform RT–PCR with total RNA from the yuc mutants (Supplementary Fig. 3).
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.
YUC1, YUC2, YUC4, and YUC6 redundantly regulate Arabidopsis growth and stature
We systematically generated all six combinations of double mutants for the four YUC genes to test for functional redundancy. Among the double mutants, yuc1yuc2, yuc1yuc6, yuc2yuc4, and yuc4yuc6 showed no obvious differences from wild type in terms of overall growth and development (data not shown). However, both yuc1yuc4 and yuc2yuc6 displayed growth and developmental defects (Fig. 2B,C). Interestingly, YUC1 and YUC4 belong to one clade, whereas YUC2 and YUC6 form another clade (Fig. 1A). This suggests that YUC1 is functionally and evolutionally closer to YUC4 and that YUC2 probably shares more overlapping functions with YUC6. Although yuc1yuc4 and yuc2yuc6 seedlings appeared normal, the rosette leaves of adult plants were slightly different from wild type (Fig. 2B). Both yuc2yuc6 and yuc1yuc4 were slightly shorter than wild type (Fig. 2C), and both double mutants showed decreased apical dominance (Fig. 2C). The yuc1yuc4 mutant was completely sterile, whereas yuc2yuc6 had dramatically reduced fertility (Fig. 2C).
The yuc1yuc2yuc6 and yuc2yuc4yuc6 triple mutants were very similar to yuc2yuc6 double mutants (Fig. 2C; data not shown). However, yuc1yuc2yuc4 and yuc1yuc4yuc6 showed phenotypes much more severe than the yuc1yuc4 double mutant (Fig. 2B,C). The yuc1yuc2yuc4 triple mutant had rosette leaves similar to those of yuc1yuc4, but with even more curvatures (Fig. 2B), whereas the rosette leaves of yuc1yuc4yuc6 were more severely affected (Fig. 2B). Compared with yuc1yuc4, the triple mutants displayed smaller statures and more extreme in decreased apical dominance (Fig. 2C). The yuc1yuc2yuc4yuc6 quadruple mutant is much smaller and has much more severe phenotypes than all of the other yuc mutant combinations (Fig. 2B,C).
The YUC1, YUC2, YUC4, and YUC6 genes have unique and overlapping functions in floral organ patterning
Both yuc2yuc6 and yuc1yuc4 are essentially sterile (Fig. 2C), but the infertile phenotypes in the two mutants were caused by defects in different parts of the flowers. Wild-type Arabidopsis flowers have four sepals, four petals, six stamens, and two fused carpels (Fig. 3A). Although all four types of organs were present in yuc2yuc6 flowers (Fig. 3B), their stamens were shorter than those of wild type (Fig. 3B). In addition, the anthers of yuc2yuc6 matured later than normal and rarely produced any pollen (Fig. 3B).
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.
Unlike yuc2yuc6, yuc1yuc4 displayed defects in all four whorls of floral organs, and no functional reproductive organs were ever observed (Fig. 3C–F). All flowers in yuc1yuc4 lacked certain floral organs (Supplementary Table 1), but the severity of defects varied among flowers. Some flowers had sepal-like organs (Fig. 3D), while other flowers totally lacked such sepal-like tissue (Fig. 3E). We observed stamen-like structures in some, but not all, flowers (Fig. 3D,E). The stamen-like structures never produced any pollen. We also observed petal-like tissue in some flowers (Fig. 3F). In some flowers (Fig. 3D), we observed two fused carpels with normal looking stigma tissue on the top. Occasionally, we observed ovules outside of the gynoecium (Fig. 3D). In other flowers, the stigma-like tissue formed a ring with a protrusion of meristem-like tissue in the center (Fig. 3E).
Like the yuc1yuc4 double mutant, yuc1yuc2yuc4 and yuc1yuc4yuc6 also displayed floral defects in all four whorls (Fig. 3G–L). Sometimes, a gynoecium-like structure grew out of the top of another gynoecium to form a spectacular structure (Fig. 3I). Compared with the double mutants, we noticed that the triple mutants made fewer flowers, and their flower structures appeared slightly smaller than the yuc1yuc4 double mutant.
The yuc1yuc2yuc4yuc6 quadruple mutant had even stronger flower phenotypes than the triple mutants described above. The inflorescence meristem was often very small, and no flower buds were visible except for some cylinder-like structures at early stages of development (Fig. 3M). Overall, the flowers of the quadruple mutant were smaller than the triple mutants. It appeared that the quadruple mutant had the ability to produce every type of the floral organ, but not every flower had all types of organs. Like the double and triple mutants, we also observed variations from flower to flower in the quadruple mutant (Fig. 3M–O).
The four YUC genes redundantly regulate venation in flowers
In wild-type flowers, vascular bundles form a continuous vascular network (Fig. 4A). The vascular bundle development in yuc2yuc6 flowers appeared normal (Fig. 4B). In the gynoecium-like structures of yuc1yuc4 flowers, the major veins are missing, and the observed vascular bundles were largely not connected to each other (Fig. 4C). The yuc1yuc2yuc4 and yuc1yuc4yuc6 triple mutants had fewer vascular bundles than the yuc1yuc4 double mutant, and in some organs there were no vascular bundles at all (Fig. 4D,E). In most cases, no veins were seen in the gynoecium-like structures in the quadruple mutant, and no vascular bundles were observed in the floral stems (Fig. 4F). It is clear that the four YUC genes are important for floral vascular development, but YUC1 and YUC4 play a more prominent role than YUC2 and YUC6.
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.
The four YUC genes are necessary for vascular development in leaves
In Arabidopsis rosette leaves, the vascular tissue is composed of a primary vein in the middle of a leaf; secondary veins originate from and connect to the primary vein, multiple tertiary veins, and higher-order veins (Fig. 4G). Overall, the vascular tissues in the yuc2yuc6 double mutant appeared to be normal (Fig. 4H). The yuc1yuc4 double mutant had fewer veins in leaves than wild type (Fig. 4I). Very few quaternary and higher-order veins presented in yuc1yuc4 (Fig. 4I). When YUC2 was inactivated in the yuc1yuc4 background, it caused a greater decrease in vein numbers (Fig. 4J). Not only were quaternary veins missing, but the yuc1yuc2yuc4 triple mutant also had fewer tertiary veins (Fig. 4J). When yuc1yuc4 was combined with yuc6, the triple mutant had even fewer secondary veins (Fig. 4K), but the veins along the edge of the leaf still formed a continuous circle. The quadruple mutant had fewer veins than the triple mutant, and the veins along the edge of the leaf no longer formed a continuous circle (Fig. 4L). The observed gradient in phenotypic strength in terms of vein numbers and continuity of the vascular strands among the yuc mutants strongly indicates that certain thresholds of local auxin concentrations, which are dependent on the YUC genes, have to be reached in order for vascular tissue formation, demonstrating that locally produced auxin is an important determinant for vascular strand formation.
Disruption of the YUC genes leads to decreased expression of the auxin reporter DR5-GUS
DR5-GUS has been widely used as an auxin reporter (Sabatini et al. 1999). In the wild-type background, GUS staining in DR5-GUS plants is observed in root tips, cotyledon tips, and newly formed leaves (Fig. 5A). The DR5-GUS-staining patterns in wild-type background presented here are the same as those previously documented (Mattsson et al. 2003). In the yuc1yuc2yuc6 mutant background, GUS staining in root tips and cotyledon tips is similar to that in wild type in terms of GUS-staining pattern and intensity (Fig. 5B). However, GUS staining in young leaves of yuc1yuc2yuc6 was much reduced (Fig. 5B). In the yuc1yuc4yuc6 background, we also observed much reduced GUS staining in young leaves, whereas staining in roots and cotyledons appeared unchanged (Fig. 5C). The reduced DR5-GUS staining was mainly observed in tissues where the YUC genes are expressed (Fig. 5A–C; Supplementary Fig. 2).
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).
Developmental defects of yuc1yuc2yuc6 and yuc1yuc4 are rescued by the bacterial auxin biosynthesis gene iaaM
To further test whether the observed developmental defects in the various loss-of-function yuc mutants were caused by auxin deficiency, we tried to rescue the yuc loss-of-function phenotypes by applying exogenous IAA (20 and 100 μM) to the mutants, but repeated applications of exogenous auxin failed to complement either yuc1yuc4 or yuc2yuc6 or other yuc mutants. It is possible that exogenous auxin may not be up-taken efficiently and accumulated to the right concentrations in cells. Therefore, we decided to mimic YUC-directed auxin production in vivo by expressing the bacterial auxin biosynthesis gene iaaM under the control of a YUC promoter. Previous studies have clearly shown that iaaM converts tryptophan to auxin once it is expressed in plants (Klee et al. 1987; Romano et al. 1995). If the phenotypes of the yuc mutants are caused by auxin deficiency, the in situ production of auxin by the iaaM gene should rescue the mutant defects. We transformed a segregating population with the iaaM gene under the control of the YUC6 promoter. The segregating population was the self-fertilized offspring from plants that were yuc1 homozygous and yuc2yuc6 heterozygous. Among the 71 T1 plants analyzed, seven plants were identified as yuc1yuc2yuc6 that also contained the iaaM gene. Like yuc2yuc6 shown above (Figs. 2C, 3B), the yuc1yuc2yuc6 triple mutant had short stamens and rarely produced any pollen (Fig. 5D), which led to essentially sterile phenotypes (Fig. 5E). The overall plant structure and stature of the iaaM transgenic plants looked like wild type. More importantly, the stamens developed normally in the iaaM transgenic lines and produced normal amounts of pollen (Fig. 5D). Furthermore, expression of the iaaM gene also rescued the infertility (Fig. 5E) and the decreased apical dominance phenotypes (Fig. 5F). The yuc1yuc2yuc6 phenotypes were also rescued by expressing the iaaM gene under the control of the YUC2 promoter (data not shown).
We also tested whether the iaaM gene can rescue the yuc1yuc4 double mutant, which displayed much more severe floral defects than the yuc1yuc2yuc6 triple mutant (Fig. 3). The yuc1yuc4 double mutant never produces any functional reproductive floral organs and is totally sterile (Figs. 2, 3). As shown in Figure 5G, expression of the iaaM gene under the control of a YUC1 promoter rescued the overall floral defects of yuc1yuc4, and the rescued plants produced functional pollen and carpel to set seeds (Fig. 5H). We never observed meristem-like tissue on top of the gynoecium in the rescued flowers, whereas such tissue is common in the yuc1yuc4 flowers (Fig. 3; Supplementary Table 1). Unlike the yuc1yuc4 flowers that never produced the right number or the normal structure of sepals, petals, and stamens (Fig. 3; Supplementary Table 1), the rescued flowers always had four sepals (Supplementary Table 2). In the rescued flowers, the structures of petals and stamens are normal looking, but the numbers varied among flowers (Supplementary Table 2). These results suggest that the amount of auxin produced by the heterologous enzyme is still not optimal. We also discovered that YUC6::iaaM could not rescue yuc1yuc4 (data not shown).
The iaaM rescue experiments clearly demonstrate that the developmental defects in yuc1yuc4 and yuc1yuc2yuc6 are caused by tissue-specific auxin deficiency and the YUC flavin monooxygenases are key enzymes for auxin biosynthesis. Our findings thus show that normal auxin functions depend on the source of auxin biosynthesis, demonstrating that regulated auxin biosynthesis by YUC flavin monooxygenases plays essential roles in Arabidopsis development.
Discussion
Auxin biosynthesis by the YUC flavin monooxygenases
Our genetic studies using both gain-of-function and loss-of-function mutants of the YUC family genes clearly demonstrate the important roles of the YUC family of flavin monooxygenase genes in auxin biosynthesis. We and others have clearly shown that overexpression of YUC1 and YUC-like genes in Arabidopsis and petunia causes auxin overproduction (Fig. 1B; Zhao et al. 2001; Marsch-Martinez et al. 2002; Tobena-Santamaria et al. 2002; Woodward et al. 2005). Like the yuc1/floozy mutant in petunia, disruption of the YUC genes in Arabidopsis also leads to defects in floral development, vascular tissue formation, and other developmental processes, indicating the essential roles of the YUC genes in plant growth and development (Figs. 2, 3, 4). We further demonstrated that inactivation of YUC genes in Arabidopsis led to reduction of GUS staining of the auxin reporter DR5-GUS (Fig. 5). Furthermore, the developmental phenotypes of the yuc mutants were rescued by expression of the bacterial auxin biosynthesis gene iaaM under the control of a corresponding YUC promoter (Fig. 5), providing unambiguous evidence that the YUC genes play a key role in auxin biosynthesis and plant development.
We have shown that the YUC genes have unique and overlapping expression patterns and their expression is temporally and spatially regulated. It becomes apparent that the location of auxin biosynthesis is critical for plant development, as we observed that different combinations of the yuc mutants display different phenotypes (Figs. 2, 3, 4). For example, the yuc2yuc6 double mutant has defects mainly in stamen development, whereas inactivation of both YUC1 and YUC4 affected the number and identity of floral organs (Fig. 3). Furthermore, we showed that the auxin reporter DR5-GUS was regulated by the YUC genes in a tissue-specific manner, demonstrating that the YUC genes provide a mechanism to produce auxin with temporal and spatial precision.
Genetic redundancy in auxin biosynthesis
It is evident that the four YUC genes (YUC1, YUC2, YUC4, and YUC6) have overlapping functions. Although the quadruple mutant had very severe developmental defects, it was not seedling or embryo lethal. Therefore we suspect that additional YUC genes have overlapping functions with the four YUC genes shown in this study. We and others have shown that at least two other YUC genes (YUC3 and YUC5) (Fig. 1A) are probably also involved in auxin biosynthesis because they caused the same phenotypes as those of yuc1D when they were overexpressed in Arabidopsis (Woodward et al. 2005; Y. Zhao, Y. Cheng, and X. Dai, unpubl.). In addition, it is known that auxin can be synthesized through the CYP79B2/B3 pathway and the tryptophan-independent pathway (Zhao et al. 2002). Construction of combinations of quintuple and sextuple yuc mutants or even higher-order mutants will be informative in defining the roles of each YUC gene in other developmental processes.
It is not understood when the duplications of the YUC genes occurred during plant species evolution. YUC genes have been found in other plant genomes including rice, maize, medicago, and petunia. Interestingly, genetic redundancy of the YUC genes was less complex in petunia, because inactivation of the YUC1 ortholog in petunia led to phenotypes similar to yuc1yuc4 in Arabidopsis (Tobena-Santamaria et al. 2002). Analysis of YUC genes in other plant species may provide clues as to how vascular plants may have evolved and how auxin may play a role during that process.
Auxin and flower development
Previous studies demonstrated that auxin signaling or polar transport mutants including pin1, pinoid, ettin, and mp have abnormal flowers, suggesting that auxin plays a critical role in flower development (Okada et al. 1991; Berleth and Jurgens 1993; Bennett et al. 1995; Sessions and Zambryski 1995). The observed floral phenotypes in the yuc mutants were very similar in many ways to the flowers of pin1, pinoid, and ettin (Okada et al. 1991; Bennett et al. 1995; Sessions et al. 1997). One similarity between the yuc flowers and the pin1/pinoid flowers is the huge variation from flower to flower in these mutants (Fig. 3). In pin1 mutants, some flowers do not have stamens, but have petals with abnormal shapes and variable numbers (Okada et al. 1991). Some pin1 flowers have a pistil-like structure without any sepals, petals, and stamens (Okada et al. 1991). Flowers of pinoid also displayed a wide range of variations (Bennett et al. 1995). As shown in Figure 3, flowers of yuc1yuc4, two triple mutants, and the quadruple mutant also had great variation. Some flowers of yuc1yuc4 were very similar to those pin1 flowers with pistil-like structures (Fig. 3D). The quadruple mutant has pin-like structures in the inflorescence apex (Fig. 3). Another similarity between the yuc mutants and the known auxin mutants (pin1, pinoid, and ettin) is that all four whorls of floral organs are affected in these mutants, indicating that auxin probably affects floral meristem. The phenotypic similarities in flowers between the yuc mutants and the known auxin mutants further support the notion that the YUC genes affect an auxin-related process, which we have shown to be auxin biosynthesis. It will be interesting to analyze mutants that combine the yuc mutants with auxin transport mutants or signaling mutants. We observed that treatment of certain yuc mutant combinations with the auxin transport inhibitor NPA totally blocked new leaf formation, a phenotype that is not observed in the yuc mutants alone or NPA treatment alone (Y. Cheng and Y. Zhao, unpubl.), suggesting that plant development is regulated by coordinated auxin biosynthesis and transport.
Unlike previous auxin mutants that only show floral organ identity defects, the yuc mutants appeared also to have the floral indeterminacy problem (Fig. 3). We often observed meristem-like tissue on the top of a gynoecium-like structure, where normally stigmatic tissue occurs (Fig. 3E,H,I,L). Occasionally, another gynoecium-like structure developed from the top of a gynoecium to produce the unprecedented floral structure (Fig. 3I), suggesting that auxin not only plays a critical role in specifying floral organ identities, but also may regulate floral meristemic cell proliferation.
Auxin and vascular tissue development
Auxin has long been suggested to play a critical role in vascular patterning. Auxin was shown to be sufficient to induce the formation of vascular strands from parenchymatic cells (Jacobs 1952), and overproduction of auxin in a whole plant increased amounts of vascular tissue (Klee et al. 1987). However, it has not been demonstrated whether auxin is also necessary for vascular tissue formation, although mutants in auxin signaling and polar transport displayed various vascular defects (Galweiler et al. 1998; Christensen et al. 2000). Compared with other auxin mutants such as pin1 and mp, the vascular defects in the yuc1yuc2yuc4yuc6 quadruple mutant were much more severe (Fig. 4). The number of veins, vascular continuity, and vascular patterns were all affected in the quadruple mutant (Fig. 4), demonstrating that auxin is essential for both vascular tissue formation and patterning.
Directional auxin flow has been suggested as a key determinant in vascular differentiation and patterning in the "canalization hypothesis" (Sachs 1981). The observed gradient in phenotypic strength in terms of vein numbers and continuity of the vascular strands among the yuc mutants strongly suggests that locally produced auxin is also an important determinant for vascular strand formation. The strong correlation between YUC gene dosage and the number of veins in leaves and flowers indicates that certain thresholds of auxin concentrations have to be reached in order for vascular tissue to form. The strong vascular phenotypes of the yuc mutants and the ordered reduction of vein numbers when YUC genes are successively compromised provides a great system for further analyzing the vascular pattern mechanism.
In summary, we have shown clearly that the YUC genes are involved in auxin biosynthesis. The YUC genes control several aspects of plant growth and development including the formation of floral organs and vascular tissues by producing auxin with temporal and spatial specifications.
Materials and methods
Materials
The T-DNA insertion mutants were obtained from either the Arabidopsis stock center at Ohio or from Dr. Joseph Ecker’s laboratory at the Salk Institute. The T-DNA mutants were genotyped as described in Tissier et al. (1999) and Alonso et al. (2003). The gene-specific primers for genotyping the T-DNA mutants were as follows: yuc1: 5'-GGTTCATGTGTTGCCAAGGGA-3' and 5'-CCTGAAGCCAAGTAGGCACGTT-3'; yuc2: 5'-CTGCATACAATCCGCTTTCGC-3' and 5'-TTCTTGCATTTTCTCGCTCTACG-3'; yuc4-1 and yuc4-2: 5'-CCCTTCTT AGACCTACTCTAC-3' and 5'-GCCCAACGTAGAATTAGC AAG-3'; yuc6: 5'-CCAGCCTTTGTATTTTCCCGT-3' and 5'-CCGGAAAAAGGGTTCTTGTCG-3'.
GUS assay and histological analysis
YUC promoters were cloned into a pBI101.3 vector, and the constructs were transformed into wild-type Arabidopsis Columbia by floral dipping (Clough and Bent 1998). The obtained transgenic plants were used for analysis of the expression patterns of each YUC gene. GUS assays were as described (Zhao et al. 2001). For whole-mount analysis of vascular structures, plant tissue was prepared as described (Berleth and Jurgens 1993), and viewed/photographed under dark-field illumination in a Leica dissecting microscope. Samples for SEM analysis were prepared according to methods described in Dinneny et al. (2004). The in situ hybridization was carried out as described in Dinneny et al. (2004).
Acknowledgments
We thank M. Yanofsky, R. Schmidt, J. Ecker, and J. Chory for helpful comments on the manuscript; Y. Liu, S. Long, D. Young, D. Chen, R. Choi, and J. Nguyen for help in genotyping the mutants; J. Long, H. Li, G. Ditta, and L. Ostergaard for some plasmids; E. York at SIO for SEM work; and J. Long, Z. Smith, L. Smith, B. Crawford, and members of the M. Yanofsky laboratory for help in sectioning and in situ hybridization. During early stages of this study, Y.Z. was funded by a NIH grant R01GM52413 to J. Chory. This work was supported by NIH grant RO1GM68631 to Y.Z.
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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.
[Keywords: Auxin; auxin biosynthesis; flavin monooxygenase; flower development; vascular development]
Received January 30, 2006; revised version accepted April 26, 2006.
Auxin is a key regulator for many aspects of plant growth and development including floral organ formation and vascular tissue patterning, but the underlying mechanisms are not well understood. It is generally believed that cell fate and organ formation in plants are determined by local auxin gradients, which are generated by PIN family proteins that control the directions of auxin flow and overall auxin distribution (Friml et al. 2002, 2003; Benkova et al. 2003; Reinhardt et al. 2003; Heisler et al. 2005; Leyser 2005). A key missing link in our understanding of auxin-regulated developmental processes is that the molecular mechanisms of auxin biosynthesis are not known. Without knowing where auxin is synthesized in plants, it is difficult to understand the exact mechanisms of auxin movements and how auxin gradients are created and maintained.
Although multiple pathways including several tryptophan-dependent routes and a tryptophan-independent pathway are proposed to synthesize auxin in plants, the molecular details and the physiological roles of each pathway are not known (Bartel 1997; Cohen et al. 2003). The only auxin biosynthesis pathway that is well defined both biochemically and genetically is the iaaM/iaaH pathway that was found in plant pathogens such as Pseudomonas and Agrobacterium, but not in plants (Kosuge et al. 1966; Comai and Kosuge 1982). The iaaM gene encodes a tryptophan-2-monooxygenase that catalyzes the conversion of tryptophan to indole-3-acetamide, which is then hydrolyzed to release indole-3-acetic acid (IAA) by the hydrolase iaaH (Kosuge et al. 1966). Genetic dissection of auxin biosynthesis in plants has proven very difficult, and no auxin-deficient mutant has ever been identified. However, there are several auxin overproduction Arabidopsis mutants including yucca (Zhao et al. 2001), sur1 (Boerjan et al. 1995; King et al. 1995), sur2 (Delarue et al. 1998), and CYP79B2 overexpression lines (Zhao et al. 2002). Both SUR1 and SUR2 are not directly involved in auxin biosynthesis, and mutations in the two genes lead to the diversion of indole-3-acetaldoxime from glucosinolate synthesis to auxin biosynthesis (Barlier et al. 2000; Mikkelsen et al. 2004). Indole-3-acetaldoxime is synthesized from tryptophan by the cytochrome P450 CYP79B2/B3, which appear not to play a critical role in plant development (Zhao et al. 2002).
We previously proposed that a flavin monooxygenase, encoded by the YUCCA1 (YUC1) gene, plays a key role in auxin biosynthesis through analysis of a dominant gain-of-function yucca mutant in Arabidopsis (renamed as yuc1D here) (Zhao et al. 2001). However, in the absence of loss-of-function mutants, it has been difficult to prove the role of YUC1 in Arabidopsis. Here we define the YUC gene family in Arabidopsis, examine the expression patterns of several YUC family members, and use reverse genetics to unequivocally show that these YUC flavin monooxygenases are involved in auxin biosynthesis. We show that disruption of certain combinations of four of the 11 predicted YUC genes (YUC1, YUC2, YUC4, YUC6) (Fig. 1A) in Arabidopsis causes dramatic developmental defects that are rescued by the bacterial auxin biosynthesis gene iaaM under the control of a corresponding YUC promoter, but not by applications of exogenous auxin. Furthermore, we show that the YUC genes are not ubiquitously expressed and inactivation of the YUC genes leads to decreased expression of an auxin reporter DR5-GUS in the cells where the YUC genes are expressed. These data provide unambiguous evidence that the loss-of-function yuc phenotypes are caused by local auxin deficiency and that the YUC flavin monooxygenases are key auxin biosynthesis enzymes. More importantly, we provide evidence demonstrating that spatially and temporally regulated auxin biosynthesis is a key determinant essential for the formation of floral organs and vascular tissues, and other developmental processes. Moreover, the temporal and spatial patterns of auxin biosynthesis now can be inferred from the expression patterns of the YUC genes, providing a necessary foundation for analysis of auxin dynamics in plants.
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.
Results
Overexpression of YUC genes leads to auxin overproduction
The Arabidopsis genome contains 10 YUC1-like genes (Fig. 1A). In this study, we focus our analysis on the roles of YUC1 and the three closest YUC1 homologs (YUC2, YUC4, and YUC6) in auxin biosynthesis and plant development (Fig. 1A). To test whether these four YUC genes function in auxin biosynthesis, we constructed transgenic Arabidopsis plants that overexpress the cDNA of each of these YUC genes under the control of the Cauliflower Mosaic Virus 35S promoter. As shown in Figure 1B, overexpression of each YUC gene led to phenotypes indistinguishable from those of yuc1D and the iaaM overexpression lines, suggesting that overexpression of each YUC gene also caused auxin overproduction. Therefore, it is likely that the YUC-like genes in this study also play a role in auxin biosynthesis because of their sequence homology with YUC1 and the observed auxin overproduction phenotypes in the overexpression lines.
Expression patterns of YUC1, YUC2, YUC4, and YUC6 in the inflorescence apex and flowers
To uncover the roles for each YUC gene in plant development, we analyzed the expression patterns of these four YUC genes by RNA in situ hybridization and by histochemistry of transgenic lines harboring promoter–GUS (-Glucuronidase) fusions. YUC4, the gene with highest sequence identity to YUC1 in Arabidopsis (Fig. 1A), shows expression patterns similar to that of YUC1 in the inflorescence apex (Fig. 1C-1, D-1). Both YUC1 and YUC4 are expressed in the apical meristems and young floral primordia (Fig. 1C-1, D-1). Initiation of flower primordia correlates well with the accumulation of YUC1 and YUC4 mRNAs (Fig. 1C-1, D-1). YUC1 is also expressed in the floral meristems and at the basal end of the young floral buds (Fig. 1C-1, C-2). It is also evident that YUC4 is expressed in the floral meristems (Fig. 1D-1). Consistent with the in situ hybridization data, the YUC1::GUS lines showed strong staining in the floral meristems and at the base of the floral organs (Fig. 1C-3). At later stages of flower development, the GUS staining of the YUC1::GUS lines is localized in discrete groups of cells in both stamens and carpels (data not shown). In the fully matured flowers, the expression of the GUS reporter in YUC1::GUS lines is completely shut down (Fig. 1C-4). The YUC4::GUS lines also show GUS staining patterns similar to those observed from the in situ hybridization (Fig. 1D-1, D-2). Significant GUS staining was observed at the base of the floral organs in young flower buds (Fig. 1D-2). In late stages of flowers, the GUS staining of YUC4::GUS lines was found predominantly in the apical regions of the carpels, stamens, and sepals (Fig. 1D-3). In mature flowers, the GUS staining was limited only to parts of the gynoecium (Fig. 1D-4). Unlike YUC1 and YUC4, YUC2 and YUC6 show very low expression in the inflorescence apex (Fig. 1E,F). However, weak staining was observed throughout the inflorescence apex including the stems of the YUC2::GUS lines (Fig. 1E-2). The YUC2::GUS lines also show staining in the young flower buds, petals, stamens, and gynoecium of young flowers (Fig. 1E-3). Staining in late stages of flowers in YUC2::GUS lines is mainly restricted to the valves of the gynoecium (Fig. 1E-3, E-4). Unlike the YUC2::GUS lines, the YUC6::GUS lines have staining mainly in stamens and pollen (Fig. 1F). The YUC genes are not ubiquitously expressed; instead, their expression is rather temporally and spatially restricted, suggesting that normal auxin functions may be dependent on the locations of auxin production.
Expression of the YUC genes in other tissues
The public Arabidopsis microarray databases (https://www.genevestigator.ethz.ch) indicate that the four YUC genes are also expressed in other tissues (Zimmermann et al. 2004) (Supplementary Fig. 1). Our promoter::GUS lines also showed that the YUC genes have distinct staining patterns in seedlings and young leaves (Supplementary Fig. 2). Strong GUS staining was observed at the base of young leaf primordia in YUC1::GUS lines, whereas GUS staining was observed in discrete spots of both basal and apical regions of young leaves in YUC4::GUS lines (Supplementary Fig. 2). We did not observe GUS staining in mature leaves in YUC1::GUS lines, but weak staining was observed in higher-order vascular strands in YUC4::GUS plants (Supplementary Fig. 2). GUS staining in YUC2::GUS lines was evident in young leaf primordia and adult leaves (Supplementary Fig. 2). No GUS staining was observed in seedlings of YUC6::GUS lines.
Identification of loss-of-function mutants for the YUC1, YUC2, YUC4, and YUC6 genes
We identified T-DNA insertion mutants for the four YUC genes from the T-DNA mutant collections (Fig. 2A; Tissier et al. 1999; Alonso et al. 2003) and carried out phenotypic analysis of the identified T-DNA mutants, but none of the mutants displayed any obvious developmental defects (data not shown). These observations are consistent with our hypothesis that there are overlapping functions among the YUC family members (Zhao et al. 2001). We did not observe any PCR products when we used primers flanking the T-DNA insertion sites to perform RT–PCR with total RNA from the yuc mutants (Supplementary Fig. 3).
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.
YUC1, YUC2, YUC4, and YUC6 redundantly regulate Arabidopsis growth and stature
We systematically generated all six combinations of double mutants for the four YUC genes to test for functional redundancy. Among the double mutants, yuc1yuc2, yuc1yuc6, yuc2yuc4, and yuc4yuc6 showed no obvious differences from wild type in terms of overall growth and development (data not shown). However, both yuc1yuc4 and yuc2yuc6 displayed growth and developmental defects (Fig. 2B,C). Interestingly, YUC1 and YUC4 belong to one clade, whereas YUC2 and YUC6 form another clade (Fig. 1A). This suggests that YUC1 is functionally and evolutionally closer to YUC4 and that YUC2 probably shares more overlapping functions with YUC6. Although yuc1yuc4 and yuc2yuc6 seedlings appeared normal, the rosette leaves of adult plants were slightly different from wild type (Fig. 2B). Both yuc2yuc6 and yuc1yuc4 were slightly shorter than wild type (Fig. 2C), and both double mutants showed decreased apical dominance (Fig. 2C). The yuc1yuc4 mutant was completely sterile, whereas yuc2yuc6 had dramatically reduced fertility (Fig. 2C).
The yuc1yuc2yuc6 and yuc2yuc4yuc6 triple mutants were very similar to yuc2yuc6 double mutants (Fig. 2C; data not shown). However, yuc1yuc2yuc4 and yuc1yuc4yuc6 showed phenotypes much more severe than the yuc1yuc4 double mutant (Fig. 2B,C). The yuc1yuc2yuc4 triple mutant had rosette leaves similar to those of yuc1yuc4, but with even more curvatures (Fig. 2B), whereas the rosette leaves of yuc1yuc4yuc6 were more severely affected (Fig. 2B). Compared with yuc1yuc4, the triple mutants displayed smaller statures and more extreme in decreased apical dominance (Fig. 2C). The yuc1yuc2yuc4yuc6 quadruple mutant is much smaller and has much more severe phenotypes than all of the other yuc mutant combinations (Fig. 2B,C).
The YUC1, YUC2, YUC4, and YUC6 genes have unique and overlapping functions in floral organ patterning
Both yuc2yuc6 and yuc1yuc4 are essentially sterile (Fig. 2C), but the infertile phenotypes in the two mutants were caused by defects in different parts of the flowers. Wild-type Arabidopsis flowers have four sepals, four petals, six stamens, and two fused carpels (Fig. 3A). Although all four types of organs were present in yuc2yuc6 flowers (Fig. 3B), their stamens were shorter than those of wild type (Fig. 3B). In addition, the anthers of yuc2yuc6 matured later than normal and rarely produced any pollen (Fig. 3B).
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.
Unlike yuc2yuc6, yuc1yuc4 displayed defects in all four whorls of floral organs, and no functional reproductive organs were ever observed (Fig. 3C–F). All flowers in yuc1yuc4 lacked certain floral organs (Supplementary Table 1), but the severity of defects varied among flowers. Some flowers had sepal-like organs (Fig. 3D), while other flowers totally lacked such sepal-like tissue (Fig. 3E). We observed stamen-like structures in some, but not all, flowers (Fig. 3D,E). The stamen-like structures never produced any pollen. We also observed petal-like tissue in some flowers (Fig. 3F). In some flowers (Fig. 3D), we observed two fused carpels with normal looking stigma tissue on the top. Occasionally, we observed ovules outside of the gynoecium (Fig. 3D). In other flowers, the stigma-like tissue formed a ring with a protrusion of meristem-like tissue in the center (Fig. 3E).
Like the yuc1yuc4 double mutant, yuc1yuc2yuc4 and yuc1yuc4yuc6 also displayed floral defects in all four whorls (Fig. 3G–L). Sometimes, a gynoecium-like structure grew out of the top of another gynoecium to form a spectacular structure (Fig. 3I). Compared with the double mutants, we noticed that the triple mutants made fewer flowers, and their flower structures appeared slightly smaller than the yuc1yuc4 double mutant.
The yuc1yuc2yuc4yuc6 quadruple mutant had even stronger flower phenotypes than the triple mutants described above. The inflorescence meristem was often very small, and no flower buds were visible except for some cylinder-like structures at early stages of development (Fig. 3M). Overall, the flowers of the quadruple mutant were smaller than the triple mutants. It appeared that the quadruple mutant had the ability to produce every type of the floral organ, but not every flower had all types of organs. Like the double and triple mutants, we also observed variations from flower to flower in the quadruple mutant (Fig. 3M–O).
The four YUC genes redundantly regulate venation in flowers
In wild-type flowers, vascular bundles form a continuous vascular network (Fig. 4A). The vascular bundle development in yuc2yuc6 flowers appeared normal (Fig. 4B). In the gynoecium-like structures of yuc1yuc4 flowers, the major veins are missing, and the observed vascular bundles were largely not connected to each other (Fig. 4C). The yuc1yuc2yuc4 and yuc1yuc4yuc6 triple mutants had fewer vascular bundles than the yuc1yuc4 double mutant, and in some organs there were no vascular bundles at all (Fig. 4D,E). In most cases, no veins were seen in the gynoecium-like structures in the quadruple mutant, and no vascular bundles were observed in the floral stems (Fig. 4F). It is clear that the four YUC genes are important for floral vascular development, but YUC1 and YUC4 play a more prominent role than YUC2 and YUC6.
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.
The four YUC genes are necessary for vascular development in leaves
In Arabidopsis rosette leaves, the vascular tissue is composed of a primary vein in the middle of a leaf; secondary veins originate from and connect to the primary vein, multiple tertiary veins, and higher-order veins (Fig. 4G). Overall, the vascular tissues in the yuc2yuc6 double mutant appeared to be normal (Fig. 4H). The yuc1yuc4 double mutant had fewer veins in leaves than wild type (Fig. 4I). Very few quaternary and higher-order veins presented in yuc1yuc4 (Fig. 4I). When YUC2 was inactivated in the yuc1yuc4 background, it caused a greater decrease in vein numbers (Fig. 4J). Not only were quaternary veins missing, but the yuc1yuc2yuc4 triple mutant also had fewer tertiary veins (Fig. 4J). When yuc1yuc4 was combined with yuc6, the triple mutant had even fewer secondary veins (Fig. 4K), but the veins along the edge of the leaf still formed a continuous circle. The quadruple mutant had fewer veins than the triple mutant, and the veins along the edge of the leaf no longer formed a continuous circle (Fig. 4L). The observed gradient in phenotypic strength in terms of vein numbers and continuity of the vascular strands among the yuc mutants strongly indicates that certain thresholds of local auxin concentrations, which are dependent on the YUC genes, have to be reached in order for vascular tissue formation, demonstrating that locally produced auxin is an important determinant for vascular strand formation.
Disruption of the YUC genes leads to decreased expression of the auxin reporter DR5-GUS
DR5-GUS has been widely used as an auxin reporter (Sabatini et al. 1999). In the wild-type background, GUS staining in DR5-GUS plants is observed in root tips, cotyledon tips, and newly formed leaves (Fig. 5A). The DR5-GUS-staining patterns in wild-type background presented here are the same as those previously documented (Mattsson et al. 2003). In the yuc1yuc2yuc6 mutant background, GUS staining in root tips and cotyledon tips is similar to that in wild type in terms of GUS-staining pattern and intensity (Fig. 5B). However, GUS staining in young leaves of yuc1yuc2yuc6 was much reduced (Fig. 5B). In the yuc1yuc4yuc6 background, we also observed much reduced GUS staining in young leaves, whereas staining in roots and cotyledons appeared unchanged (Fig. 5C). The reduced DR5-GUS staining was mainly observed in tissues where the YUC genes are expressed (Fig. 5A–C; Supplementary Fig. 2).
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).
Developmental defects of yuc1yuc2yuc6 and yuc1yuc4 are rescued by the bacterial auxin biosynthesis gene iaaM
To further test whether the observed developmental defects in the various loss-of-function yuc mutants were caused by auxin deficiency, we tried to rescue the yuc loss-of-function phenotypes by applying exogenous IAA (20 and 100 μM) to the mutants, but repeated applications of exogenous auxin failed to complement either yuc1yuc4 or yuc2yuc6 or other yuc mutants. It is possible that exogenous auxin may not be up-taken efficiently and accumulated to the right concentrations in cells. Therefore, we decided to mimic YUC-directed auxin production in vivo by expressing the bacterial auxin biosynthesis gene iaaM under the control of a YUC promoter. Previous studies have clearly shown that iaaM converts tryptophan to auxin once it is expressed in plants (Klee et al. 1987; Romano et al. 1995). If the phenotypes of the yuc mutants are caused by auxin deficiency, the in situ production of auxin by the iaaM gene should rescue the mutant defects. We transformed a segregating population with the iaaM gene under the control of the YUC6 promoter. The segregating population was the self-fertilized offspring from plants that were yuc1 homozygous and yuc2yuc6 heterozygous. Among the 71 T1 plants analyzed, seven plants were identified as yuc1yuc2yuc6 that also contained the iaaM gene. Like yuc2yuc6 shown above (Figs. 2C, 3B), the yuc1yuc2yuc6 triple mutant had short stamens and rarely produced any pollen (Fig. 5D), which led to essentially sterile phenotypes (Fig. 5E). The overall plant structure and stature of the iaaM transgenic plants looked like wild type. More importantly, the stamens developed normally in the iaaM transgenic lines and produced normal amounts of pollen (Fig. 5D). Furthermore, expression of the iaaM gene also rescued the infertility (Fig. 5E) and the decreased apical dominance phenotypes (Fig. 5F). The yuc1yuc2yuc6 phenotypes were also rescued by expressing the iaaM gene under the control of the YUC2 promoter (data not shown).
We also tested whether the iaaM gene can rescue the yuc1yuc4 double mutant, which displayed much more severe floral defects than the yuc1yuc2yuc6 triple mutant (Fig. 3). The yuc1yuc4 double mutant never produces any functional reproductive floral organs and is totally sterile (Figs. 2, 3). As shown in Figure 5G, expression of the iaaM gene under the control of a YUC1 promoter rescued the overall floral defects of yuc1yuc4, and the rescued plants produced functional pollen and carpel to set seeds (Fig. 5H). We never observed meristem-like tissue on top of the gynoecium in the rescued flowers, whereas such tissue is common in the yuc1yuc4 flowers (Fig. 3; Supplementary Table 1). Unlike the yuc1yuc4 flowers that never produced the right number or the normal structure of sepals, petals, and stamens (Fig. 3; Supplementary Table 1), the rescued flowers always had four sepals (Supplementary Table 2). In the rescued flowers, the structures of petals and stamens are normal looking, but the numbers varied among flowers (Supplementary Table 2). These results suggest that the amount of auxin produced by the heterologous enzyme is still not optimal. We also discovered that YUC6::iaaM could not rescue yuc1yuc4 (data not shown).
The iaaM rescue experiments clearly demonstrate that the developmental defects in yuc1yuc4 and yuc1yuc2yuc6 are caused by tissue-specific auxin deficiency and the YUC flavin monooxygenases are key enzymes for auxin biosynthesis. Our findings thus show that normal auxin functions depend on the source of auxin biosynthesis, demonstrating that regulated auxin biosynthesis by YUC flavin monooxygenases plays essential roles in Arabidopsis development.
Discussion
Auxin biosynthesis by the YUC flavin monooxygenases
Our genetic studies using both gain-of-function and loss-of-function mutants of the YUC family genes clearly demonstrate the important roles of the YUC family of flavin monooxygenase genes in auxin biosynthesis. We and others have clearly shown that overexpression of YUC1 and YUC-like genes in Arabidopsis and petunia causes auxin overproduction (Fig. 1B; Zhao et al. 2001; Marsch-Martinez et al. 2002; Tobena-Santamaria et al. 2002; Woodward et al. 2005). Like the yuc1/floozy mutant in petunia, disruption of the YUC genes in Arabidopsis also leads to defects in floral development, vascular tissue formation, and other developmental processes, indicating the essential roles of the YUC genes in plant growth and development (Figs. 2, 3, 4). We further demonstrated that inactivation of YUC genes in Arabidopsis led to reduction of GUS staining of the auxin reporter DR5-GUS (Fig. 5). Furthermore, the developmental phenotypes of the yuc mutants were rescued by expression of the bacterial auxin biosynthesis gene iaaM under the control of a corresponding YUC promoter (Fig. 5), providing unambiguous evidence that the YUC genes play a key role in auxin biosynthesis and plant development.
We have shown that the YUC genes have unique and overlapping expression patterns and their expression is temporally and spatially regulated. It becomes apparent that the location of auxin biosynthesis is critical for plant development, as we observed that different combinations of the yuc mutants display different phenotypes (Figs. 2, 3, 4). For example, the yuc2yuc6 double mutant has defects mainly in stamen development, whereas inactivation of both YUC1 and YUC4 affected the number and identity of floral organs (Fig. 3). Furthermore, we showed that the auxin reporter DR5-GUS was regulated by the YUC genes in a tissue-specific manner, demonstrating that the YUC genes provide a mechanism to produce auxin with temporal and spatial precision.
Genetic redundancy in auxin biosynthesis
It is evident that the four YUC genes (YUC1, YUC2, YUC4, and YUC6) have overlapping functions. Although the quadruple mutant had very severe developmental defects, it was not seedling or embryo lethal. Therefore we suspect that additional YUC genes have overlapping functions with the four YUC genes shown in this study. We and others have shown that at least two other YUC genes (YUC3 and YUC5) (Fig. 1A) are probably also involved in auxin biosynthesis because they caused the same phenotypes as those of yuc1D when they were overexpressed in Arabidopsis (Woodward et al. 2005; Y. Zhao, Y. Cheng, and X. Dai, unpubl.). In addition, it is known that auxin can be synthesized through the CYP79B2/B3 pathway and the tryptophan-independent pathway (Zhao et al. 2002). Construction of combinations of quintuple and sextuple yuc mutants or even higher-order mutants will be informative in defining the roles of each YUC gene in other developmental processes.
It is not understood when the duplications of the YUC genes occurred during plant species evolution. YUC genes have been found in other plant genomes including rice, maize, medicago, and petunia. Interestingly, genetic redundancy of the YUC genes was less complex in petunia, because inactivation of the YUC1 ortholog in petunia led to phenotypes similar to yuc1yuc4 in Arabidopsis (Tobena-Santamaria et al. 2002). Analysis of YUC genes in other plant species may provide clues as to how vascular plants may have evolved and how auxin may play a role during that process.
Auxin and flower development
Previous studies demonstrated that auxin signaling or polar transport mutants including pin1, pinoid, ettin, and mp have abnormal flowers, suggesting that auxin plays a critical role in flower development (Okada et al. 1991; Berleth and Jurgens 1993; Bennett et al. 1995; Sessions and Zambryski 1995). The observed floral phenotypes in the yuc mutants were very similar in many ways to the flowers of pin1, pinoid, and ettin (Okada et al. 1991; Bennett et al. 1995; Sessions et al. 1997). One similarity between the yuc flowers and the pin1/pinoid flowers is the huge variation from flower to flower in these mutants (Fig. 3). In pin1 mutants, some flowers do not have stamens, but have petals with abnormal shapes and variable numbers (Okada et al. 1991). Some pin1 flowers have a pistil-like structure without any sepals, petals, and stamens (Okada et al. 1991). Flowers of pinoid also displayed a wide range of variations (Bennett et al. 1995). As shown in Figure 3, flowers of yuc1yuc4, two triple mutants, and the quadruple mutant also had great variation. Some flowers of yuc1yuc4 were very similar to those pin1 flowers with pistil-like structures (Fig. 3D). The quadruple mutant has pin-like structures in the inflorescence apex (Fig. 3). Another similarity between the yuc mutants and the known auxin mutants (pin1, pinoid, and ettin) is that all four whorls of floral organs are affected in these mutants, indicating that auxin probably affects floral meristem. The phenotypic similarities in flowers between the yuc mutants and the known auxin mutants further support the notion that the YUC genes affect an auxin-related process, which we have shown to be auxin biosynthesis. It will be interesting to analyze mutants that combine the yuc mutants with auxin transport mutants or signaling mutants. We observed that treatment of certain yuc mutant combinations with the auxin transport inhibitor NPA totally blocked new leaf formation, a phenotype that is not observed in the yuc mutants alone or NPA treatment alone (Y. Cheng and Y. Zhao, unpubl.), suggesting that plant development is regulated by coordinated auxin biosynthesis and transport.
Unlike previous auxin mutants that only show floral organ identity defects, the yuc mutants appeared also to have the floral indeterminacy problem (Fig. 3). We often observed meristem-like tissue on the top of a gynoecium-like structure, where normally stigmatic tissue occurs (Fig. 3E,H,I,L). Occasionally, another gynoecium-like structure developed from the top of a gynoecium to produce the unprecedented floral structure (Fig. 3I), suggesting that auxin not only plays a critical role in specifying floral organ identities, but also may regulate floral meristemic cell proliferation.
Auxin and vascular tissue development
Auxin has long been suggested to play a critical role in vascular patterning. Auxin was shown to be sufficient to induce the formation of vascular strands from parenchymatic cells (Jacobs 1952), and overproduction of auxin in a whole plant increased amounts of vascular tissue (Klee et al. 1987). However, it has not been demonstrated whether auxin is also necessary for vascular tissue formation, although mutants in auxin signaling and polar transport displayed various vascular defects (Galweiler et al. 1998; Christensen et al. 2000). Compared with other auxin mutants such as pin1 and mp, the vascular defects in the yuc1yuc2yuc4yuc6 quadruple mutant were much more severe (Fig. 4). The number of veins, vascular continuity, and vascular patterns were all affected in the quadruple mutant (Fig. 4), demonstrating that auxin is essential for both vascular tissue formation and patterning.
Directional auxin flow has been suggested as a key determinant in vascular differentiation and patterning in the "canalization hypothesis" (Sachs 1981). The observed gradient in phenotypic strength in terms of vein numbers and continuity of the vascular strands among the yuc mutants strongly suggests that locally produced auxin is also an important determinant for vascular strand formation. The strong correlation between YUC gene dosage and the number of veins in leaves and flowers indicates that certain thresholds of auxin concentrations have to be reached in order for vascular tissue to form. The strong vascular phenotypes of the yuc mutants and the ordered reduction of vein numbers when YUC genes are successively compromised provides a great system for further analyzing the vascular pattern mechanism.
In summary, we have shown clearly that the YUC genes are involved in auxin biosynthesis. The YUC genes control several aspects of plant growth and development including the formation of floral organs and vascular tissues by producing auxin with temporal and spatial specifications.
Materials and methods
Materials
The T-DNA insertion mutants were obtained from either the Arabidopsis stock center at Ohio or from Dr. Joseph Ecker’s laboratory at the Salk Institute. The T-DNA mutants were genotyped as described in Tissier et al. (1999) and Alonso et al. (2003). The gene-specific primers for genotyping the T-DNA mutants were as follows: yuc1: 5'-GGTTCATGTGTTGCCAAGGGA-3' and 5'-CCTGAAGCCAAGTAGGCACGTT-3'; yuc2: 5'-CTGCATACAATCCGCTTTCGC-3' and 5'-TTCTTGCATTTTCTCGCTCTACG-3'; yuc4-1 and yuc4-2: 5'-CCCTTCTT AGACCTACTCTAC-3' and 5'-GCCCAACGTAGAATTAGC AAG-3'; yuc6: 5'-CCAGCCTTTGTATTTTCCCGT-3' and 5'-CCGGAAAAAGGGTTCTTGTCG-3'.
GUS assay and histological analysis
YUC promoters were cloned into a pBI101.3 vector, and the constructs were transformed into wild-type Arabidopsis Columbia by floral dipping (Clough and Bent 1998). The obtained transgenic plants were used for analysis of the expression patterns of each YUC gene. GUS assays were as described (Zhao et al. 2001). For whole-mount analysis of vascular structures, plant tissue was prepared as described (Berleth and Jurgens 1993), and viewed/photographed under dark-field illumination in a Leica dissecting microscope. Samples for SEM analysis were prepared according to methods described in Dinneny et al. (2004). The in situ hybridization was carried out as described in Dinneny et al. (2004).
Acknowledgments
We thank M. Yanofsky, R. Schmidt, J. Ecker, and J. Chory for helpful comments on the manuscript; Y. Liu, S. Long, D. Young, D. Chen, R. Choi, and J. Nguyen for help in genotyping the mutants; J. Long, H. Li, G. Ditta, and L. Ostergaard for some plasmids; E. York at SIO for SEM work; and J. Long, Z. Smith, L. Smith, B. Crawford, and members of the M. Yanofsky laboratory for help in sectioning and in situ hybridization. During early stages of this study, Y.Z. was funded by a NIH grant R01GM52413 to J. Chory. This work was supported by NIH grant RO1GM68631 to Y.Z.
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