The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis

Genetic and molecular studies in Arabidopsis and Antirrhinum have led to the proposal of the ABC model for control of floral organ identity (Haughn and Somerville, 1988; Coen and Meyerowitz, 1991; Meyerowitz et al., 1991; Ma, 1994; Weigel and Meyerowitz, 1994; Yanofsky, 1995; Ma and dePamphilis, 2000). Particularly, the Arabidopsis B function genes APETALA3 (AP3) and PISTILLATA (PI) are required to specify petal and stamen identities (Bowman et al., 1989; Hill and Lord, 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Both AP3 and PI are expressed in specific regions of the floral meristem prior to the initiation of petal and stamen primordia (Jack et al., 1992; Goto and Meyerowitz, 1994) at stage 3 of Arabidopsis flower development (Smyth et al., 1990). The stable spatial pattern of AP3 and PI expression is directly correlated with the control of organ identity, as further supported by the fact that ectopic expression of both AP3 and PI leads to the formation of ectopic petals and stamens (Jack et al., 1994; Krizek and Meyerowitz, 1996).

The Arabidopsis floral meristem identity gene LEAFY (LFY) is required for normal levels of AP3 and PI expression (Weigel and Meyerowitz, 1993), consistent with the lack of petals and stamens in severe lfy mutants (Schultz and Haughn, 1991; Huala and Sussex, 1992; Weigel et al., 1992). However, flowers of weak lfy mutants, such as lfy-5, can still produce petals and stamens. Another Arabidopsis gene, UNUSUAL FLORAL ORGANS (UFO), also plays a role in controlling floral meristem development and the B function (Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995; Samach et al., 1999). Moreover, the activation of AP3 expression by LFY requires UFO (Lee et al., 1997; Parcy et al., 1998), although how UFO interacts with LFY is not known. The SUPERMAN (SUP, or FLO10) gene can also regulate B function in Arabidopsis and is expressed shortly after the onset of AP3 and PI expression (Schultz et al., 1991; Bowman et al., 1992; Sakai et al., 1995; Jacobsen and Meyerowitz, 1997; Sakai et al., 2000). In addition, AP3 and PI are expressed ectopically in the sup mutant floral meristems (Bowman et al., 1992; Sakai et al., 1995). These observations led to the hypothesis that SUP acts to maintain the boundary between whorl 3 and 4, possibly by controlling differential cell division in different domains of the floral meristems (Sakai et al., 1995; Sakai et al., 2000).

The cell division cycle is regulated by both the synthesis and degradation of key regulatory proteins. Proteolysis is essential for many normal cellular functions, but its role in plant development is not clear. A major pathway for protein degradation is the ubiquitin-dependent pathway by the 26S proteosome (Ciechanover et al., 2000). Ubiquitin is a highly conserved small protein that is covalently attached to proteins through a three-step process requiring the E1, E2, and E3 enzymes (Jentsch and Pyrowolakis, 2000). Whereas the E1 and E2 enzymes are rather non-specific, the E3 ubiquitin ligase confers substrate specificity. The SCF E3 ubiquitin ligase complexes are named after the three subunits: SKP1, cullin (CDC53 in yeast), and one of the F-box containing proteins, which are the substrate specificity factors (Feldman et al., 1997; Skowyra et al., 1997; Peters, 1998; Craig and Tyers, 1999). The yeast SKP1 gene is essential for the mitotic cell cycle (Bai et al., 1996; Connelly and Hieter, 1996).

The Arabidopsis UFO protein (Ingram et al., 1995) contains an F-box, suggesting that it may be a subunit of a SCF ubiquitin ligase. Furthermore, UFO and its Antirrhinum homologue FIM have been found using yeast two-hybrid assays to interact with homologues of the yeast and human SKP1 proteins, including the Arabidopsis ASK1 gene product (Ingram et al., 1997; Samach et al., 1999). ASK1 was shown to be expressed in dividing cells, including meristems and floral organ primordia (Porat et al., 1998), consistent with a potential role in cell division. We have previously isolated a male-sterile transposon insertion, ask1-1, in the ASK1 gene (Yang et al., 1999). The ask1-1 mutant also has mild defects during vegetative and reproductive development (Zhao et al., 1999). Furthermore, some ask1-1 flowers exhibit abnormality in petals and stamens, including reduced number and size of petals and reduced stamen filament lengths, suggesting a weak defect in B function (Zhao et al., 1999). We further showed that ASK1 and UFO interact genetically with each other, consistent with the observed interaction using the yeast two-hybrid method (Samach et al., 1999). These results support the hypothesis that UFO and ASK1 may be subunits of a SCF ubiquitin ligase required for normal Arabidopsis flower development, particular for regulating B functions.

To further investigate the function of ASK1, we have constructed additional double and triple mutants between ask1 and other mutations, including ufo, ap3, pi, sup, and lfy. Our results support the idea that ASK1 interacts with UFO to regulate B function genes AP3 and PI. To more directly test this idea, we have performed RNA in situ hybridization experiments and found that indeed ask1 mutation can cause a reduction of AP3 and PI expression when LFY gene function is reduced by a weak mutation. We propose that ASK1 and UFO together control AP3 and PI expression via a negative regulator of these genes. In addition, we describe results indicating a role for ASK1 in regulating the number of floral organ primordia, and discuss their implications.

The wild type and mutants used were in the Landsberg erecta (Ler) backrgound. The ask1-1 mutant was isolated as a male sterile mutant and it has a Ds transposon insertion in the middle of the protein-coding region upstream of a highly conserved domain (Yang et al., 1999; Zhao et al., 1999). The other mutants have been described previously: ap3-1 and pi-1 (Bowman et al., 1989), ap3-3 (Jack et al., 1992), lfy-5 and lfy-6 (Weigel et al., 1992), ufo-2 and ufo-6 (Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995), sup-1 (Bowman et al., 1992). Seeds were sown onto Metro-Mix 360 (Scotts-Sierra Horticultural Products Co., Maryville, OH), incubated for 4 days at 4°C and then grown at 23°C with long-day cycles (16 hours light and 8 hours dark).

All single mutants used for phenotypic comparison were derived from self-pollination of either homozygous (e.g., ufo-2) or heterozygous (e.g., ap3-3/+) plants. To construct double and triple mutants, the male sterile and female fertile ask1-1 mutant (Yang et al., 1999) was used as the female in crosses whenever possible. For crosses with ap3-1, ap3-3, or pi-1, pollen from ask1-1/+ heterozygous plants was used. We had previously generated partially fertile ufo-2/ufo-2 ask1-1/+ plants (Zhao et al., 1999), which were used as male for crosses to generate triple mutants with ap3-3 and pi-1 mutations. In addition, ufo-2/ufo-2 ask1-1/ask1-1 plants were pollinated with pollen from sup-1, lfy-5, or lfy-6/+ plants to generate the sup-1 ufo-2, lfy-5 ufo-2, and lfy-6 ufo-2 double mutants and the triple mutants. The ask1-1 Ds insertion confers kanamycin resistance, allowing the selection on MS kanamycin plates for double heterozygous F₁ plants from crosses using ask1-1/+ plants. For crosses with lfy-6/+, F₂ seeds from multiple F₁ plants were harvested and tested for segregation of each relevant single mutants. All F₁ plants that were doubly or triply heterozygous were normal.

The ask1-1 mutant has a shorter stature than normal (Zhao et al., 1999); this characteristic is unique among the mutants studied here and was used to identify candidate ask1-1 homozygous plants. The ask1-1 mutant has many morphologically normal flowers and can be easily distinguished from the ap3-1, ap3-3, pi-1, sup-1, and lfy-6 mutants; furthermore, sup-1 is male fertile but usually female sterile. The ufo-2, ufo-6, and lfy-5 mutants also have mild floral phenotypes, but are male and female fertile, are of normal height and lack normal flowers, unlike ask1-1 plants. Therefore, all known single floral mutants can be distinguished from ask1-1 based on a combination of plant stature, floral morphology and fertility. Furthermore, the ASK1 allele was confirmed by a PCR product using the ASK1 gene-specific primers oMC221 (5′-AAG GTG ATC GAG TAT TGC AAG AG-3′) and oMC 383 (5′-GAA GAT AGT CAT GAT TCA TGA AG-3′); the ask1-1 mutant allele was verified by the oMC221 primer and the Ds-specific primer Ds 5-2 (5′-CGT TCC GTT TTC GTT TTT TAC C-3′).

The double mutants with ask1-1 and another mutation were identified using phenotypes and PCR tests for either ASK1 or ask1-1 alleles. For example, among the F₂ plants from the cross between ap3-3 and the ask1-1/+ heterozygote, in addition to the ask1-1 and ap3-3 single mutants, a rare class of mutants produced ap3-3 like flowers and was as short as ask1-1 single mutant. These candidate double mutant plants were confirmed to be homozygous for the ask1-1 allele by PCR. The pi-1 ask1-1 and lfy-6 ask1-1 double mutants were similarly identified. The ap3-1 ask1-1 and lfy-5 ask1-1 double mutants were more easily recognized because they had more severe floral phenotypes than either single mutant of the respective crosses. Additional lfy-5 ask1-1 plants were obtained from progeny of lfy-5/lfy-5 ask1-1/+ plants. The sup-1 ask1-1 double mutant had abnormal carpels, similar to sup-1; at the same time, it was also male sterile as is ask1-1. These double mutants were nevertheless confirmed by using PCR. Statistical analyses indicate that the segregation data can be accepted according to χ² tests (Table 1).

To identify triple mutants with ufo-2, ask1-1 and either ap3-3, pi-1, sup-1, lfy-5 or lfy-6, plants with floral phenotypes similar to, or more severe than those of the third mutant, were first confirmed as being ask1-1/ask1-1 by PCR, and then tested for UFO genotype using PCR. Three primers were designed based on the UFO genomic sequence. Two of them were the same except for 3′-end nucleotides, one matched the wild-type UFO sequence (oMC396: 5′-TGG TAA GAT GGT TTA CGT GC-3′) and the other matched the sequence of the ufo-2 allele (oMC 397 5′-TGG TAA GAT GGT TTA CGT GT-3′). The third primer (oMC410: 5′ TAA CCA CCG GTG TAG TAA GC 3′) was used with either of the other two primers. Both PCR experiments were performed with each candidate plant, and the UFO genotype of the plants was determined by comparing the relative amount of these two PCR products (Li et al., 1999). The sup-1 ufo-2, lfy-5 ufo-2 and lfy-6 ufo-2 double mutants were identified similarly among ASK1/ASK1 plants.

Light microscopic images were recorded digitally using a Nikon dissecting microscope and Optronics camera, and processed using Photoshop. Additional flowers were examined using a Nikon dissecting microscope. Samples for scanning electron microscopy were fixed, dried, dissected and coated, and then the specimens were examined as previously described (Bowman et al., 1989) using a JSM 5400 (JEOL USA, Peabody, MA).

RNA in situ hybridizations were performed on wild-type and mutant floral sections as previously described (Drews et al., 1991; Flanagan and Ma, 1994). The AP3 and PI antisense and sense probes were synthesized using in vitro transcription reactions with the pD793 and pcPINX plasmids as templates, respectively (Jack et al., 1992; Goto and Meyerowitz, 1994).

We compared the floral phenotypes of the wild type (Fig.1A), single mutants, the double and triple mutants of ask1-1 with the following mutations: ap3-1, ap3-3, pi-1, ufo-2, ufo-6, and sup-1 (Fig. 1; Table 2). Our results on the single mutants and the ufo-2 ask1-1 double mutant are in agreement with previous reports (Bowman et al., 1989; Bowman et al., 1991; Bowman et al., 1992; Jack et al., 1992; Goto and Meyerowitz, 1994; Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995; Zhao et al., 1999).

The ask1-1 mutant flowers sometimes show a reduction of petal number and petal size (Fig.1B), reduced stamen filament length, and petal/anther chimeras (Zhao et al., 1999). The ufo-2 flower (Fig. 1C) has abnormal floral organs interior to whorl one, including ectopic sepals, petals, stamens, carpels, filaments, or chimeric organs (Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995). The ufo-2 ask1-1 double mutant flower (Fig. 1D) had a similar phenotype to the ufo-2 single mutant (Zhao et al., 1999).

The ufo-6 weak mutant has slightly affected petals and stamens (Levin and Meyerowitz, 1995), with variable flower phenotypes consisting of chimeric petals and reduction of petal number and size (Fig. 1E,F). To obtain further support for an interaction between UFO and ASK1, we constructed the ufo-6 ask1-1 double mutant. In some ufo-6 ask1-1 flowers, petals were similar to those in ask1-1, but carpelloid organs and filaments were often found (Fig. 1G). Furthermore, the ufo-6 ask1-1 could sometimes produce flowers with a phenotype very similar to that of ufo-2 (Fig. 1H). The enhancement of the ufo-6 phenotype by ask1-1 supports a genetic interaction between these two genes.

Because both UFO and ASK1 affect organ identity in whorls two and three, we wanted to analyze double and triple mutants with ap3 mutations. The ap3-1 mutant is a temperature sensitive weak mutant (Bowman et al., 1989); we observed that at 23°C ap3-1 flowers had sepals in whorl two and stamens, staminoid or carpelloid organs in whorl three (Fig. 1I). In contrast, ap3-1 ask1-1 flowers (Fig. 1J,K) had filaments or carpelloid organs interior to whorl one but no stamens at all, similar to the strong ap3-3 mutant flower which also has filaments, carpelloid organs and/or carpels (Fig. 1L; Bowman et al., 1989; Bowman et al., 1991; Jack et al., 1992).

We had previously generated the ap3-3ask1-1 double mutant (Zhao et al., 1999) and briefly reported its flower phenotype, which is similar to that of ap3-3 in terms of organ type. We show here that ap3-3 ask1-1 flowers have fewer floral organs than the ap3-3 flower (Table 2). Specifically, ap3-3 ask1-1 flowers had fewer than four sepals interior to whorl one (Fig. 1M; Table 2), which was significantly different from ap3-3 (T value=24.41). Also sepals in some ap3-3 ask1-1 flowers were small (Fig. 1N). Interior to whorl one, the number of filaments in ap3-3 ask1-1 flowers was not significantly different from that in ap3-3 flowers (Table 2, T value=0.49). The total organ number in ap3-3 ask1-1 flowers was significantly reduced compared to that in ap3-3 flowers (Table 2, T value=10.87).

To test for genetic interaction between AP3, UFO and ASK1, we also examined the ap3-3 ufo-2 ask1-1 triple mutant and found that it was similar to ap3-3 ask1-1, except that the organ number was perhaps further reduced slightly. In some ap3-3 ufo-2 ask1-1 flowers, we found only one or two sepals and no filament interior to whorl one (Fig.1O). Some ap3-3 ufo-2 ask1-1 flowers did not form any organs at all between whorls one and four (Fig. 1P). These results indicate that the effect of ap3-3 ask1-1 and ap3-3 ufo-2 ask1-1 on flowers were more severe than ap3-3 alone in terms of organ number.

We also characterized double and triple mutants involving pi-1, which causes the formation of abnormal organs interior to whorl one, similar to ap3-3 (Bowman et al., 1989; Bowman et al., 1991) (Fig. 1Q). Although the pi-1 ask1-1 flowers showed a similar phenotype to that of pi-1 flowers, the double mutant flowers had fewer floral organs interior to whorl one than pi-1 flowers (Fig. 1R; Table 2). Most pi-1 ask1-1 flowers had fewer than four sepals interior to whorl one, which was significantly different from that of pi-1 (T value=23.02). In addition, the number of filaments in the double mutant flower was significantly greater than that of the pi-1 single mutant (T value=7.31), but the total number of floral organs in the pi-1 ask1-1 flower was significantly smaller than in the pi-1 flower (Table 2, T value=4.68).

Although ap3-3 ask1-1 and pi-1 ask1-1 flowers had similar number of sepals interior to whorl one, pi-1 ask1-1 flowers produced more filaments or filament-like organs than ap3-3 ask1-1 flowers (Table 2). The pi-1 ufo-2 ask1-1 triple mutant flower seemed to have a slightly more severe phenotype than either the pi-1 ask1-1 double mutant or the pi-1 single mutant in terms of the total floral organ number (Fig. 1S). In addition, pi-1 ufo-2 ask1-1 flowers made fewer filaments than pi-1 ask1-1 flowers (Fig. 1S and data not shown).

We also analyzed double and triple mutants with the sup-1 mutation, which causes the production of flowers with extra stamens interior to whorl two and a reduced carpelloid organ in the center (Bowman et al., 1992; Fig. 1T). Most sup-1 flowers had about ten stamens and a dramatically reduced carpelloid organ (Bowman et al., 1992) (Table 2). However, the sup-1 ask1-1 double mutant flowers usually produced approximately seven stamens, significantly different from sup-1 (Table 2, T value=13.24). In addition, sup-1 ask1-1 flowers had a larger carpelloid organ in the center than did sup-1 (Fig. 1U; Table 2, T value=18.83). About 10% of the double mutant flowers could even make a normal pistil in the fourth whorl (Fig. 1V). Furthermore, sup-1 ask1-1 flowers had reduced number and size of petals, short stamen filaments and sterile anthers, similar to the ask1-1 flowers.

Compared to the sup-1 mutant, the sup-1 ufo-2 flowers also had a reduction in stamen number and an enlargement of the carpelloid organs, similar to the sup-1 ask1-1 flowers (Fig. 1W; Levin and Meyerowitz, 1995). But the sup-1 ufo-2 flower had fewer petals and stamens than the sup-1 ask1-1 flowers and was male fertile. The sup-1 ufo-2 ask1-1 triple mutant flower was slightly more similar to sup-1 ufo-2 than sup-1 ask1-1 flowers (Fig. 1X). Compared with the two double mutants, the number of petals and stamens in the triple mutant was even smaller and the central carpelloid organ was slightly larger.

Our observations with mature flowers described above indicate that ask1-1 and ufo-2 mutations alone or together caused a reduction of organ number in the ap3-3, pi-1, and sup-1 backgrounds. We were interested to determine when the effect of ask1-1 and ufo-2 can be detected during flower development and whether these mutations affect floral organ primordium initiation; therefore, we examined early floral development of the double and triple mutants using scanning electron microscopy.

First we compared flower development between ap3-1 single and ap3-1 ask1-1 double mutants. There was no detectable difference in the inflorescence meristem and early floral primordia before stage 5 between ap3-1 and ap3-1 ask1-1 mutants (not shown). At stage 6, the ap3-1 floral bud (Fig. 2D) showed sepal primordia in whorl two and stamen primordia in whorl three, but the size was smaller than wild type (Fig. 2A; Bowman et al., 1989). Although the stage-6 ap3-1 ask1-1 bud (Fig. 2F) had four sepal primordia interior to whorl one, similar to that of ap3-1, it lacked the characteristic stamen primordia. In addition, some peripheral regions of the central carpel primordia were enlarged (Fig. 2F). In the stage-7 ap3-1 ask1-1 floral bud, the carpel primordia continued to enlarge, but there were no stamen primordia (Fig. 2G). The ap3-1 floral bud at about stage 10 could form stamen primordia (Fig. 2E) which were smaller than the wild-type ones (Fig. 2C). However, the ap3-1 ask1-1 floral bud at stage 10 only produced sepals, filaments or carpelloid organs (Fig. 2H), without any stamens.

Similar to the ap3-1 ask1-1 floral bud (Fig. 2F), the ap3-3 floral bud at stage 6 had four sepal primordia interior to whorl one and enlarged carpel primordia in the center (Fig. 2I). In contrast, the ap3-3 ask1-1 floral bud at stage 6 formed fewer sepal primordia than the ap3-3 single mutant, even though both of them could produce similarly enlarged carpel primordia (Fig. 2K). At about stage 10, the ap3-3 bud usually had sepals, filaments, or carpelloid organs interior to whorl one (Fig. 2J), again similar to ap3-1 ask1-1 buds (Fig. 2H). In comparison, ap3-3 ask1-1 flowers produced fewer floral organs interior to whorl one than ap3-3 flowers. Some ap3-3 ask1-1 flowers had fewer than four sepals and no filament structure (Fig. 2L). Some ap3-3 ask1-1 flowers had no sepals or sepal-like organs (not shown). The ap3-3 ufo-2 ask1-1 triple mutant flower was similar to the ap3-3 ask1-1 flower, except that the triple mutant flower had slightly fewer floral organs than the ap3-3 ask1-1 flower (Fig. 2M,N). Our observations indicate that the ask1-1 mutation could enhance the ap3-1 phenotype and the ask1-1 and ufo-2 mutations reduced the number of floral organ primordia in the ap3-3 background.

We also examined the early floral morphology of double and triple mutants with the pi-1 mutation. We observed that at stage 6 both pi-1 single (Fig. 2O) and pi-1 ask1-1 double mutant (Fig. 2Q) floral buds formed enlarged carpel primordia at the center; however, the pi-1 ask1-1 bud at this stage showed fewer sepal primordia than the pi-1 bud. At a later stage the pi-1 ask1-1 flower produced fewer floral organs than pi-1(Fig. 2P), sometimes lacking sepals interior to whorl one (Fig. 2R). The pi-1 ufo-2 ask1-1 floral buds at stage 6 (not shown) and approximately stage 10 (Fig. 2S) had fewer floral organ primordia than the pi-1 ask1-1 floral buds. Therefore, the ask1-1 and ufo-2 mutations also caused a reduction of floral organ primordia in the pi-1 background.

Finally, we analyzed the early floral morphology of double and triple mutants with the sup-1 mutation. Before stage 5, there was no detectable difference between the sup-1 single and sup-1 ask1-1 double mutant floral buds (not shown). The stage-6 sup-1 floral bud (Fig. 2T) formed six stamen primordia in whorl three and began to form more stamen primordia. The gynoecium primordium at the center was shorter than in the wild type. At stage 9, the sup-1 flower produced more than six stamens and no obvious carpel structures (Fig. 2U). The sup-1 ask1-1 bud at stage 6 (Fig. 2V) was similar to that of sup-1. In some late sup-1 ask1-1 flowers, we found six stamens and fused carpels (Fig. 2W), which was similar to the wild type at this stage (Fig. 1C). The sup-1 ufo-2 ask1-1 triple mutant flower had a similar floral phenotype to that of sup-1 ask1-1, but produced fewer stamens and a slightly larger carpel-like structure (Fig. 2X).

The analyses of these double and triple mutants indicate that ask1-1 and ufo-2 mutations cause a reduction of organ primordium initiation interior to whorl one in the ap3, pi and sup mutant backgrounds. In addition, the combination of both ask1-1 and ufo-2 mutations results in a slightly greater reduction in organ initiation.

Because lfy mutations affect floral organ identity in a way consistent with a defect in B function (Schultz and Haughn, 1991; Huala and Sussex, 1992; Weigel et al., 1992), we tested for possible interaction between ASK1, UFO and LFY by comparing the floral phenotypes of single, double and triple mutants. The strong lfy-6 mutant flowers only had leaf-like and carpel-like organs (Fig. 3A; Weigel et al., 1992). Flowers of the lfy-6 ask1-1and lfy-6 ufo-2 double mutants (Fig. 3B, C) and the lfy-6 ufo-2 ask1-1triple mutant flower (Fig. 3D) had similar phenotypes, suggesting that ask1-1 and ufo-2 mutations have no effect in the lfy-6 background.

We then analyzed double and triple mutants between ask1-1, ufo-2 and the weak allele lfy-5. Flowers of the weak lfy-5 mutant had well-developed petals, stamens and carpels (3.0 petals, 2.7 stamens, and 2.2 carpels, n=30; Fig. 3E) (Weigel et al., 1992). In contrast, the lfy-5 ask1-1 double mutant flower had a much more severe phenotype than that of lfy-5, and closely resembled that of lfy-6. Most of the lfy-5 ask1-1 flowers only produced leaf-like and carpel-like organs (7.2 and 2.9, respectively, n=30; Fig. 3F). Nevetheless, we occasionally found that the lfy-5 ask1-1 flower had stamen or stamen-like organs (0.2, n=30), which were never found in the lfy-6 flower. The lfy-5 ufo-2 flower was similar to that of lfy-5 ask1-1 (Fig. 3G); futhermore, the lfy-5 ufo-2 ask1-1triple mutant had no detectable difference from lfy-6 (Fig. 3H). These results suggest that the combination of a partial loss of LFY function (lfy-5) and ask1-1 and ufo-2 mutations can cause a similar floral defect to the complete loss of LFY function (lfy-6).

We have examined the early floral morphology of single, double and triple mutants carrying lfy mutations. In the lfy-6 floral bud at about stage 6, the first four leaf-like primordia formed a whorl, but the other leaf-like primordia developed in a spiral pattern (Fig. 4A). At later stages lfy-6 flowers produced leaf-like organs with branched trichomes (Fig. 4B,C). Both in early and late stages, lfy-6 ask1-1and lfy-6 ufo-2 ask1- 1 floral buds had similar phenotypes to that of the lfy-6 single mutant (data not shown). Therefore, the ask1-1 and ufo-2 mutations did not affect early flower development in the lfy-6 background.

We further compared early flower development in the weak lfy-5 mutant and corresponding double and triple mutants. The stage 6 lfy-5 floral bud (Fig. 4D) had stamen primordia that were nearly normal in size, but their number was reduced compared to the wild type. In addition, we observed nearly normal carpel primordia at the center of the lfy-5 floral bud (Fig. 4D). The late lfy-5 flower clearly showed well developed petals, stamens and carpels (Fig. 4E,F). However, the development of lfy-5 ask1-1 flower was quite different from lfy-5 flowers. The lfy-5 ask1-1 floral bud at about stage 6 (Fig. 4G) produced leaf-like primordia in a spiral pattern, similar to the lfy-6. The leaf-like primordia eventually developed into leaf-like organs (Fig. 4H,I). The lfy-5 ufo-2 ask1-1 flowers had similar phenotypes to that of lfy-5 ask1-1, and were not detectably different from the lfy-6 flower (data not shown). We conclude that when LFY function is reduced, ASK1 and UFO function are important for the specification of floral organ primordia identities and phyllotaxy.

Our results from phenotypic studies suggest that ASK1 and UFO interact with B function genes and LFY genetically. It is known that LFY and UFO positively regulate the expression of B function genes AP3 and PI. Therefore, it is possible that ASK1 also contributes to the positive regulation of AP3 and PI expression. To test this idea, we performed RNA in situ hybridization to determine AP3 and PI expression in wild-type, single and double mutant inflorescence sections. Our results for AP3 expression in wild-type and lfy-6 and PI expression in the wild type were in agreement with previous findings (Jack et al., 1992; Weigel and Meyerowitz, 1993; Goto and Meyerowitz, 1994).

The onset of AP3 expression has been shown to occur at stage 3 in the wild-type floral meristem (Fig. 5A; Jack et al., 1992). During stages 5-8, AP3 was present in petal and stamen primordia at a high level. After stage 9, the level of AP3 mRNA was reduced, but still detectable. The ask1-1 flower showed a normal AP3 expression pattern (Fig. 5B), but the expression level in some mutant flowers was slightly reduced (not shown). In the lfy-5 flower, AP3 mRNA was clearly detectable in stage-3 to -5 floral meristems, but the level was considerably lower than normal (Fig. 5C,D). After stage 6, the AP3 mRNA was present in the lfy-5 bud at a slightly lower level than either the wild-type or the ask1-1 mutant buds (data not shown). AP3 mRNA was not detectable in most lfy-6 flowers and only occasionally found at the base in some lfy-6 flowers (Fig. 5E,F). The lfy-5 ask1-1 flowers showed an AP3 expression pattern very similar to those of lfy-6. In most lfy-5 ask1-1 flowers, the AP3 mRNA was not detectable, although a very limited amount of AP3 signal was observed at the base of some flowers (Fig. 5G,H).

The PI mRNA was first detected in the wild-type stage 3 bud and it remained present at a high level in the developing petals and stamens (Goto and Meyerowitz, 1994) (Fig. 5I). The ask1-1 (Fig. 5J) and lfy-5 (Fig. 5K,L) flowers exhibited similar PI expression pattern to that of the wild-type flower, but the PI mRNA level in some ask1-1 and lfy-5 flowers was slightly lower than that of wild type. In contrast, the PI expression was not detectable in most areas of lfy-6 flowers, except for a limited amount of PI signal at the base of some flowers (Fig. 5M,N). Similarly, PI mRNA was not detectable in most regions of lfy-5 ask1-1 flowers, with only a small amount of PI signal at the base of some flowers (Fig. 5O,P). The results from the AP3 and PI in situ hybridization experiments indicate that the ask1-1 and lfy-5 mutations together cause a much more severe reduction of AP3 and PI mRNA levels and domains than either single mutations.

ASK1 and UFO both affect petal and stamen identities in whorls two and three, respectively, and interact with each other genetically; furthermore, the ASK1 and UFO proteins have been shown to interact physically (Samach et al., 1999; Zhao et al., 1999). These findings suggested that ASK1 and UFO may act together to regulate B function. In this report we show that ask1-1 can further enhance the floral organ identity phenotype of ap3-1. At the same time, our observations indicate that ask1-1 does not enhance the floral organ identity defects of ap3-3 and pi-1 mutations. Moreover, triple mutants with ask1-1, ufo-2 and ap3 or pi mutations, showed similar organ identity phenotypes to ap3-3 and pi-1 mutants. It has been argued that although null alleles of genes in the same genetic pathway should not enhance each other’s phenotypes, partially functional mutations and/or mutations in functionally redundant genes could enhance each other’s phenotypes (Martienssen and Irish, 1999). Therefore, our double and triple mutant phenotypes support the idea that ASK1 and UFO may function in the same regulatory network that requires AP3 and PI gene functions, i.e., the B function of the ABC model for the specification of floral organ identity.

However, ask1-1 and ufo-2 single mutants, even the ufo-2 ask1-1 double mutant, are less severe than the ap3-3 and pi-1 mutants. This may be due to functional redundancy because Arabidopsis has additional SKP1 homologues (ASK2∼ASK9, Gray et al., 1999; Samach et al., 1999; other ASKs, GenBank/Arabidopsis Sequencing Initiative). Three of these genes, ASK2, ASK3 and ASK18, have 70% or more amino acid sequence identity to ASK1 and might have similar functions to ASK1 in flower development. Similarly, UFO is an F-box containing protein; there are dozens, if not hundreds, of putative F-box-containing proteins predicted by the Arabidopsis genome sequencing project (Arabidopsis Sequencing Initiative). The potential existence of functionally similar genes to both ASK1 and UFO could explain why mutations in these genes cause less severe floral phenotypes. This is supported by the observed physical interaction between UFO and ASK2 (Samach et al., 1999) and by the observation that ASK2 has a similar expression pattern in early floral buds to that of ASK1 (D. Z. and H. M., unpublished data).

The ask1-1 mutation enhances the phenotype of the weak lfy-5 mutant, but not that of the strong lfy-6 mutant, suggesting that ASK1 likely functions in the same regulatory pathway as LFY. Previous studies showed that LFY is a positive regulator of AP3 and PI expression and that UFO is an important co-regulator of LFY (Lee et al., 1997; Parcy et al., 1998). Our results suggest that ASK1 may also be a co-regulator of LFY for the activation of AP3 and PI expression. Indeed, this hypothesis was further supported by our findings that the expression of both the AP3 and PI genes was reduced to a much greater extent by the combination of ask1-1 and lfy-5 mutations than by either mutation alone. Furthermore, the reduction of AP3 and PI expression in the lfy-5 ask1-1 double mutant flowers was very similar to that in lfy-6, a presumed null allele. This result and the fact that lfy-6 single, lfy-6 ask1-1 and lfy-6 ufo-2 double, and lfy-6 ask1-1 ufo-2 triple mutants all have nearly identical floral phenotypes suggest that the regulation of B function by ASK1 and UFO requires LFY function.

AP3 and PI have slightly different domains of expression initially, with the PI expression domain closer to the center of the floral meristem (Jack et al., 1992; Goto and Meyerowitz, 1994). In addition, it was shown that the ufo-1 mutation causes a reduction of early AP3 expression, but not PI expression (Samach et al., 1999). This and the fact that the 35S-AP3, but not the 35S-PI, transgene could rescue the ufo-1 mutant phenotype in whorl three led to the idea that UFO positively regulates AP3 expression, but not that of PI (Samach et al., 1999). However, the 35S-PI transgene also did not rescue the pi-1 mutant in whorl three, suggesting that the transgene might not provide enough PI function (Krizek and Meyerowitz, 1996; Samach et al., 1999). In addition, the lack of reduction of PI expression in the ufo-1 mutant could be explained by a possible functional redundancy of UFO and other F-box proteins. Furthermore, the observation that PI is expressed throughout 35S-LFY 35S-UFO seedlings strongly supports the idea that LFY and UFO also positively regulate PI expression (Honma and Goto, 2000). Our results support the hypothesis that ASK1 positively regulates the expression of both AP3 and PI with LFY, as well as UFO.

We observed that the ap3-3 and pi-1 mutations caused an reduction of organ number interior to whorl one, consistent with earlier studies (Bowman et al., 1989; Bowman et al., 1991; Jack et al., 1992; Goto and Meyerowitz, 1994). Furthermore, ectopic expression of AP3 and PI resulted in extra whorls of stamens (Krizek and Meyerowitz, 1996). Therefore, in addition to their roles in specifying organ identity, the AP3 and PI genes also promote cell proliferation, especially near the center of the floral meristem (Jack et al., 1992; Krizek and Meyerowitz, 1996). In addition, it was previously observed that sup-1 mutants have reduced floral meristem determinacy, resulting in additional whorl(s) of stamens (Schultz et al., 1991; Bowman et al., 1992; Sakai et al., 2000). The ectopic expression of AP3 and PI in sup-1 floral meristem also supports a role for AP3 and PI in cell proliferation and initiation of floral organ primordia.

We showed previously that the ask1-1 mutant flowers had a slightly reduced number of petals and a nearly normal number of other organs (Zhao et al., 1999; Table 2). ASK1 is homologous to the yeast SKP1 gene, which is an essential regulator of cell division and encodes a subunit of the SCF ubiquitin ligase. Therefore, ASK1 may also regulate cell proliferation during flower development. We further observed that flowers of the sup-1 ask1-1 double mutant and the sup-1 ufo-2 ask1-1 triple mutant had fewer stamens or stamen-like organs and more carpels than the sup-1 single mutant. Therefore, the increased whorl-three cell proliferation in sup-1 mutant requires ASK1 and UFO functions. We found that sup-1 ask1-1 flowers had a nearly normal number of carpels, more than the sup-1 mutant; therefore, relative to sup-1, reduction of whorl three is balanced by an increase in whorl four.

Because the ask1-1 mutation can cause a reduction in AP3 and PI expression in the lfy-5 background, the opposite of the effect of the sup-1 mutation, part of ASK1 function in regulating cell proliferation may be mediated by AP3 and PI. Furthermore, the ask1-1 mutation could enhance the phenotype of ap3-3 or pi-1 mutants in the reduction of floral organ number interior to whorl one, particularly the number of sepal or sepal-like organs (Table 2). Therefore, the ASK1 and AP3/PI genes seem to have redundant functions in regulating cell proliferation in this region of the flower. This suggests that part of ASK1’s function in cell proliferation is independent of AP3 and PI.

UFO is an F-box containing protein and ASK1 is a homologue of the yeast and human SKP1 protein (Ingram et al., 1995; Yang et al., 1999). Both SKP1 and F-box containing proteins are subunits of the SCF ubiquitin ligase complex, suggesting that ASK1 and UFO might be components of a SCF complex that facilitates the degradation of a negative regulator of B function gene expression. For example, ASK1 and UFO may control the level of a negative modulator (X) of LFY protein activity (Fig. 6A). When the ASK1 and UFO proteins are both functional, the level of X is low, and the LFY protein is fully active. However, if the ASK or UFO gene is mutated, then X is present at an increased level. When LFY protein is normal, the effect of X is minor, but when LFY protein activity is reduced by mutations such as lfy-5, then the negative effect of X becomes much more obvious. Alternatively, ASK1 and UFO may regulate a direct repressor (Y) of AP3 and PI expression, whereas LFY is an activator of these genes (Fig. 6B). In this case, we need to postulate that when LFY is fully functional, the presence of Y, due to ask1 or ufo mutations, cannot reduce AP3 and PI expression substantially. In contrast, when LFY function is reduced by the lfy-5 mutation, then Y repression of AP3 and PI becomes effective. In either model, ASK1 and UFO could also interact with other partners to regulate AP3 and PI expression; nevertheless, mutant phenotypes and RNA expression analysis suggest that ASK1 and UFO are the primary players in the proposed network of regulators.

These possibilities could be tested by analyzing cis elements in AP3 and PI promoters that mediate regulation by LFY and UFO/ASK1. If the first scenario is true, then the same elements should mediate the effects of both LFY and UFO/ASK1 because X regulates LFY activity. If the second situation is true, then Y could bind to a different site from the LFY-binding site(s) in the AP3 and PI upstream regions. Promoter studies of AP3 revealed that a region from −328 to the transcriptional start seems to mediate the effect of UFO, and the −1500 to −300 region of the PI promoter mediates the effects of LFY and UFO (Hill et al., 1998; Honma and Goto, 2000). Furthermore, within the −328 to 0 region of the AP3 promoter, there are three putative sites (CArG boxes) for binding by MADS proteins; mutational analysis suggests that two of these mediate activation, whereas the third (CArG3) mediates repression of AP3 (Hill et al., 1998; Tilly et al., 1998). Because the precise sites mediating LFY and UFO regulation were not mapped, further analysis is required to distinguish the above models.

We have shown here that the ASK1 gene cooperates with LFY to activate AP3 and PI expression, and it plays an important role in regulating floral organ primordia in whorls two and three. Our results also suggest that UFO also participates in these regulatory processes. The fact that ASK1 and UFO are both putative subunits of the SCF ubiquitin ligase suggests these proteins may regulate the level of other regulatory proteins that control cell division and/or transcription. These results support the idea that regulatory proteolysis can play important roles in controlling flower development.

We thank J. Bowman, E. Meyerowitz, D. Weigel, and the Ohio State University Arabidopsis Stock Center for providing mutant seeds, T. Jack and E. Meyerowitz for the AP3 and PI probes. We also thank D. Weigel and the anonymous reviewers for helpful discussion and critical reading of this manuscript. In addition, we thank Y. Hu for technical assistance, R. Walsh for assistance with electron microscopy, A. Omeis for plant care, and E. Harris and M. Henry for assistance with plant work. This work is supported by a grant from the National Science Foundation (MCB-9896340), and by Funds from the Department of Biology and the Life Sciences Consortium at Pennsylvania State University.