Anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ligase, plays a critical role in cell cycle control, but the functional characterization of each subunit has not yet been completed. To investigate the function of APC1 in Arabidopsis, we analyzed four mutant alleles of APC1, and found that mutation in APC1 resulted in significantly reduced plant fertility, accumulation of cyclin B, and disrupted auxin distribution in embryos. The three mutant alleles apc1–1, apc1–2 and apc1–3 shared variable defects in female gametogenesis including degradation, abnormal nuclear number, and disrupted polarity of nuclei in the embryo sac as well as in embryogenesis, in which embryos were arrested at multiple stages. All of these defects are similar to those previously identified in apc4. The mutant apc1–4, in which the T-DNA was inserted after the transmembrane domain at the C-terminus, showed much more severe phenotypes; that is, most of the ovules were arrested at the one-nucleate female gametophyte stage (stage FG1). In the apc1 apc4 double mutants, the fertility was further reduced by one-third in apc1–1/+ apc4–1/+, and in some cases no ovules even survived in siliques of apc1–4/+ apc4–1/+. Our data thus suggest that APC1, an essential component of APC/C, plays a synergistic role with APC4 both in female gametogenesis and in embryogenesis.
APC1 is the largest subunit of the anaphase-promoting complex/cyclosome (APC/C) (Capron et al. 2003a), and is conserved among different species from bacteria to higher plants and animals (Figure S1). In Arabidopsis, APC1 is encoded by a single copy gene, At5g05560 (Capron et al. 2003a). AtAPC1 contains 33 exons and 32 introns (Figure 1A), and encodes a protein of 1,678 amino acid residues with two predicted transmembrane domains at its C-terminus (Figure 1B). To investigate the function of APC1 in Arabidopsis, we used four independent mutant alleles of APC1, that is, apc1–1, apc1–2, apc1–3 and apc1–4. apc1–1 was identified from the activation tagging mutant collection in this laboratory (Qin et al. 2003), and apc1–2, apc1–3 and apc1–4 were requested from the Arabidopsis Biological Resource Center (ABRC). These four apc1 mutants had a T-DNA inserted in the 3rd, 4th, 11th and 32nd exon of the APC1 gene, respectively (Figure 1A). Genotyping analysis of the apc1/+ progeny (n > 80 per line) identified no homozygotes from any of these four apc1 alleles. To find out whether the mutation of APC1 caused gametophyte sterility and/or embryo lethality, we conducted genetic analyses. In apc1–1/+, the segregation ratios of progeny indicated by phosphinothricin-resistant (PPTR) seedlings to PPT-sensitive (PPTS) seedlings on PPT selective medium remained stable around 1.1–1.2 in three continuous generations (Table 1, Table S1), which apparently deviates from the typical Mendelian segregation ratio of 3:1 (Drews and Yadegari 2002). This suggested that apc1–1 was a gametophytic mutant. Reciprocal crosses between apc1–1/+ plants and wild-type (WT) plants showed that, when apc1–1/+ pistils were pollinated with pollen from WT plants, the F1 progeny had a PPTR:PPTS ratio of 0.26:1 (184/699, Table 1), and when WT pistils were pollinated with mutant pollen, the F1 progeny had a ratio of 1.01:1 (938/929, Table 1). Similar results were observed in apc1–2/+ and apc1–3/+ (data not shown). These results indicated that the transmission of the female apc1 gamete was greatly reduced, whereas the male transmission was normal. Further microscopic observation revealed that all four apc1 mutant alleles had siliques with reduced seed sets (Figure 1C), that is, about 55% in apc1–1/+, apc1–2/+ and apc1–3/+, and approximately 35% in apc1–4/+ (Table 2). Two types of abnormalities were observed in siliques from the four apc1/+ heterozygotes: undeveloped ovules and aborted seeds (Figure 1C, Table 2). The percentage of either defect was consistently similar in the siliques from apc1–1/+, apc1–2/+ and apc1–3/+. The siliques from the apc1–4/+ mutants had a much higher percentage of undeveloped ovules than the other three apc1/+ mutant alleles (Figure 1C, Table 2).
Table 1. The progeny segregation ratio from selfing and reciprocal crosses of apc1–1 plants
PPT R: PPTS a
aThe T-DNA in apc1–1/+ carries a herbicide (phosphinothricin, PPT) resistance gene. Seedlings were cultured on 1/2 Murashige & Skoog (MS) medium with 10 μg/mL PPT. R, resistant; S, sensitive.
bn, number of individual lines analyzed.
1.19:1 (3231/2708, n= 20)b
0.26:1 (184/699, n= 7)
1.01:1 (938/929, n= 8)
Table 2. Seed development in four apc1 alleles
Aberrant seeds (n)a
Total (n) b
Percentage of aberrant seeds (%)c
aTotal of the aberrant seeds.
bTotal seeds observed.
cPercentage of aberrant seeds. R = a/b.
The apc1 mutation caused multiple developmental defects in female gametogenesis
To further uncover the underlying mechanisms of reduced female transmission observed in apc1/+, we investigated the process of female gametogenesis of the mutants via Confocal Laser Scanning Microscopy (CLSM). In WT Arabidopsis, female gametogenesis undergoes a synchronized process, FG1–FG7, to form a mature embryo sac (Figure 2A–F) (Christensen et al. 1997; Liu and Qu 2008). In apc1/+ plants, although the synchrony of female gametogenesis was not affected (Table S2), developmental defects, in particular abnormal nuclei behavior, were observed at almost all the developmental stages of the embryo sac (Figure 2G–L, Table S3). Similar to the defects observed in apc4/+ (Wang et al. 2012), three categories of phenotypes were mainly observed in the ovules from apc1–1/+, apc1–2/+ and apc1–3/+: degradation of the nuclei or embryo sac (6.8% in apc1–1/+) (Figure 2G), mis-positioned nuclei (2.3% in apc1–1/+) (Figure 2H, I) and abnormal nuclear number (5.8% in apc1–1/+) (Figure 2J, K). A small portion of ovules were arrested at stage FG1 (0.8%) in apc1–1/+ (Figure 2L). The phenotypes in apc1–4/+ were much more severe than those in the other three alleles. In addition to the three categories of defects, the dominant abnormality in apc1–4/+ was that about 41.5% of the ovules were arrested at stage FG1 (Figure 2L). The total percentage of the abnormal ovules in apc1/+ observed by CLSM was slightly lower than that of those examined by stereomicroscopy (Table 2). One explanation for this difference could be that some early aborted embryos were possibly grouped into the undeveloped ovules due to their high similarity in morphology under stereomicroscopy.
The apc1 mutation resulted in aberrant embryogenesis, possibly involving maternal effects
To further clarify the aborted seeds phenotypes observed in apc1/+ plants (Table 2), we examined embryogenesis by differential interference contrast microscopy (DIC). In WT plants, a fertilized zygote goes through a series of developmental stages to form a mature embryo in an ordered, polarized and symmetrical manner (Figure 3A–F) (Goldberg et al. 1994; Xin et al. 2012). In apc1/+ plants, about 15% of the embryos were arrested at variable stages: 3.7% of the embryos at early stages (postfertilization-proembryo-globular stage, Figure 3G–I), 8.5% at the heart to torpedo stage (Figure 3J–L) and 3.0% at the late stages (walking stick to mature stage, Figure 3M–O). Some of these aborted embryos also showed disordered polarity and disrupted symmetry, probably due to aberrant cell division or cell growth (Figure 3H–O). Given the fact that the APC4 subunit was involved in maternal effects (Wang et al. 2012), we performed reciprocal crosses between apc1/+ and WT plants. Similar to those of apc4/+, the crossed siliques using apc1–1/+ as female showed a comparable percentage of aborted seeds to that observed in selfing siliques (Figure 4, Table 3). Similar to apc1-1/+, pollen from WT plants could not rescue the seed abortion phenotypes in apc1-4/+ siliques either (Table S4). These results indicate that, similar to APC4, APC1 could possibly regulate embryogenesis via maternal effects.
Table 3. Seed development in siliques from reciprocal crosses between apc1–1/+ and wild-type (WT)
Female × male
Seed phenotypes (%)a
Total (n) d
aSeed phenotypes include undeveloped ovules and aborted seeds.
bUndeveloped ovules and aborted embryos before (including) proembryo stage.
cAborted embryos from the globular stage to the mature stage.
dTotal seeds examined.
Wild-type × wild-type
Ovules in apc1/+ plants displayed accumulated cyclin B-GUS protein
To investigate whether loss-of-function of APC1 abolishes the function of APC/C as an ubiquitin ligase, we examined the protein level of a well-known substrate of APC/C, cyclin B (King et al. 1995; Harper et al. 2002), in apc1/+, by crossing cyclin B-GUS reporter line to apc1–1/+ plants. The inflorescences of the cyclin B-GUS reporter line and the apc1/+ plants crossed with the cyclin B-GUS line were collected for a histochemical β-glucuronidase (GUS) assay. In the cyclin B-GUS reporter lines, no significant GUS signal was detected in inflorescence (Figure 5A). In apc1–1/+, however, an obvious GUS signal was detected in the ovules at about stage 12 (FG7) and in the pistils at stage 15 (proembryo) (Figure 5B), indicating a compromised function of APC/C in apc1 mutants. Interestingly, the accumulation of cyclin B-GUS protein in apc1–1/+ (Figure 5B) was more obvious than that observed in apc4–1/+ (Wang et al. 2012), suggesting a more important role of APC1 in maintaining the function of APC/C in cell cycle regulation.
Auxin distribution during embryogenesis was disrupted in apc1/+ plants
Since embryo development was greatly impaired in apc1/+ (Figure 3), we wondered whether auxin distribution was affected in the mutant, because auxin plays an essential role in regulating embryogenesis (Cooke et al. 1993; Liu et al. 1993). We crossed the DR5rev::GFP reporter line, which carries the auxin responsive promoter DR5 and is used to examine the spatial distribution of auxin (Sabatini et al. 1999; Benkova et al. 2003), with the apc1/+ plants. Since the majority of the aberrant embryos were arrested at the heart or torpedo stage (8.5% out of 15.2%), we primarily analyzed the embryos at these two stages. In WT embryos at the heart or torpedo stages, the GFP signal was mainly observed in primordial tips of cotyledons, provascular cells, and in the hypophysis/root meristem in a symmetrical pattern (Figure 6A, B). However, in apc1/+ embryos, the pattern of the GFP signal was significantly disrupted. It was asymmetrically strengthened in provascular cells (Figure 6C), and partially detected in hypophysis (Figure 6D) and cotyledons (Figure 6E). The disrupted auxin distribution could be observed both in embryos with aberrant morphology (Figure 6C) and in embryos with normal morphology (Figure 6D), suggesting that the disrupted auxin distribution occurred before the morphological abnormalities of the embryos emerged.
Plant fertility was greatly reduced in apc1 apc4 double mutants
Both APC1 and APC4 are APC/C subunits, and loss-of-function of either subunit leads to disrupted female gametogenesis and embryogenesis. To further investigate the relationship between APC1 and APC4, we generated apc1 apc4 double loss-of-function mutants. Two apc1 alleles, apc1–1/+ and apc1–4/+, were used to cross with apc4–1/+ plants. In the vegetative stage, no significant difference was observed among apc1–1/+, apc4–1/+ and apc1–1/+ apc4–1/+ plants. In the reproductive stage, however, the double mutant had significantly shortened siliques compared to the two single mutants (Figure S2). We further examined the seed development in siliques from single mutants and double mutants, and found that the seed sets in double mutant siliques were significantly reduced compared with those in the single mutants (Figure 7, Table 4). The seed set in apc1–1/+ apc4–1/+ was reduced to 32.6% (Figure 7E, Table 4), whereas in apc1–4/+ apc4–1/+, the seed set was significantly reduced to 15% (Figure 7F, Table 4). In some severe cases in apc1–4/+ apc4–1/+, almost all of the ovules were aborted (Figure 7G). Taken together, plant fertility was much more reduced in apc1 apc4 double mutants than in single mutants, suggesting that APC1 possibly plays a synergistic role to APC4 in Arabidopsis female gametogenesis and embryogenesis.
Table 4. Seed development in siliques from apc1, apc4 and apc1 apc4 mutants
Seed set (%)
Although the full-length cDNA of APC1 was not cloned due to the large size of this gene, the fact that the three mutant alleles apc1–1, apc1–2 and apc1–3, shared similar developmental defects in gametogenesis and embryogenesis indicates that APC1 is the functional gene. The T-DNA insertion sites in apc1–1, apc1–2 and apc1–3 were relatively close to each other in the 5′ region of APC1, while in apc1–4, the T-DNA insertion site was in the 32nd exon close to the 3′ region of APC1 (Figure 1A). The large difference between the T-DNA insertion sites in the first three apc1 alleles and in apc1–4 may account for why apc1–4 exhibited more severe phenotypes in female gametogenesis than the other three mutant alleles in apc1–4. It is reasonable to speculate that there may be some undefined domains/motifs at the C-terminus that are potentially critical for the proper function of APC1. This needs to be clarified in future studies. In yeast and mammals APC/C, APC1 functions as a scaffold module (van Leuken et al. 2008; McLean et al. 2011), and APC4 serves as one of the two connectors (van Leuken et al. 2008; Wang et al. 2012). In Arabidopsis, it is interesting to note that the three apc1 mutant alleles shared similar phenotypes during female gametogenesis and embryogenesis to those observed in apc4 (Wang et al. 2012). The female gametogenesis of either of the three apc1 mutant alleles or of the apc4 mutants was disrupted with abnormal nuclei degradation, nuclear number and positions at variable stages, whereas the embryogenesis was arrested at all developmental stages with distorted auxin distribution. These results suggest that APC1 and APC4 may be involved in similar biological processes. The genetic evidence that the double mutants of either combination exhibited much more severe phenotypes than either of the single mutants suggests that APC1 and APC4 play synergistic roles in regulating female gametogenesis and embryogenesis. This probably reflects the potential roles of these two proteins in the APC/C complex. APC4 is reported as one of the two connectors; loss-of-function of APC4 results in partial abruption of the connection of other sub-complexes. Since the scaffold is still functioning, at least part of the APC/C activity should be maintained, and thus defects in female gametogenesis and embryogenesis are not completely uncontrollable. If the scaffold protein APC1 is disrupted, severe defects in female gametogenesis and embryogenesis can be expected. The normal function of connectors would somehow keep the structure of APC/C and part of its activity. However, if both the scaffold protein and the connector were disrupted, the activity of APC/C would be difficult to maintain. This is probably the reason why the double mutants had much more severe phenotypes.
Another possibility is that APC1, a large but flexible scaffold protein, may interact with different proteins. In yeast and mammals, functional APC/C is regulated by different kinds of proteins, including activators (CDC20, CCS52A, CCS52A2 and CCS52B in plants) (Fülöp et al. 2005), inhibitors (SAMBA (Eloy et al. 2012), UVI4 (Hase et al. 2006; Heyman et al. 2011) and OSD1/UVI4-Like/GIGAS (Iwata et al. 2011; Cromer et al. 2012)), and substrates (e.g., cyclin B). Most of the known APC/C subunits are predicted to interact with a number of proteins (http://thebiogrid.org/, accessed 27 September 2012). For example, both APC1 and APC4 were shown to interact with the activators of CDC20.1, FZR2 (CCS52A1), CCS52A2 and FZR3 (CCS52B), and the inhibitor of OSD1/UVI4-Like/GIGAS. In addition, the two proteins were found to interact with other subunits, including APC2, HBT/CDC27b, APC7, APC8/CDC23 and APC10. However, only APC1, rather than APC4, was able to interact with APC11 and the negative regulator UVI4. The similar protein interaction network of APC1 and APC4 may account for the similar phenotypes shared by these two mutants, and may reflect the close position of these two proteins in similar structural roles in maintaining and stabilizing the conformation of the APC/C complex (Thornton et al. 2006; van Leuken et al. 2008; Herzog et al. 2009). Taken together, our results indicate that the stable structure of APC/C is critical for its proper activity, in which both the scaffold protein APC1 and the connector APC4 play an essential and synergistic role in maintaining the APC/C structure. Functional characterization of all the APC/C subunits and elucidation of their structures will eventually facilitate our understanding of substrate recognition, degradation, stability and activity regulation of the APC/C complex in Arabidopsis.
All of the Arabidopsis plants used in this study, including the wild-type (WT) plants, transgenic plants and T-DNA insertion mutants, were in the Columbia ecotype. apc1–1 was generated in our lab, while the other three apc1 alleles, apc1–2, apc1–3 and apc1–4, were obtained from the Arabidopsis Biological Resource Center (ABRC) with the stock names of SALK_059826, SALK_152651 and SALK_111190, respectively. The T-DNA insertion sites in these alleles were confirmed by sequencing the flanking sequence. Primers used for genotypic analysis are listed in Table S5. Plants were grown as previously reported (Qin et al. 2005; Luo et al. 2012). Seeds of apc1–1 were sowed on 1/2 Murashige & Skoog (MS, Sigma-Aldrich, St. Louis, MO, USA) medium with 10 μg/mL PPT (SIGMA45520, Sigma-Aldrich), and cultured under 16 h light/8 h dark conditions for 7–10 d before segregation ratio analysis.
Female gametogenesis observation
Confocal laser scanning microscopy (CLSM) observation for female gametogenesis was performed as previously described by Liu et al. (2008). A LEICA TCS SPE Confocal Microscope was used (Leica, Germany).
Embryo development observation
Fresh embryos were isolated from siliques with tweezers (Fontax, Switzerland, 5 INOX) and cleared by Hoyer's solution (Berleth and Jurgens 1993) from minutes to hours depending on the embryo stages. Cleared embryos were then observed by differential interference contrast (DIC) microscopy under a microscope (BX51, Olympus, Tokyo, Japan) equipped with a CCD camera (SPOT Diagnostic Instruments, Sterling Heights, MI, USA). For fluorescent signal detection in live embryos, fresh embryos were mounted in 5% glycerol and observed under a LEICA TCS SPE Confocal Microscope (Leica, Wetzlar, Germany).
Histochemical GUS Assays
The histochemical GUS assays were performed as previously described by Qin et al. (2005). The inflorescences were pre-fixed and dipped into the GUS staining solution and then incubated in 37°C for approximately 8 h.
The accession number for Arabidopsis APC1 in TAIR is At5g05560. Amino acid sequences of APC1s from different species are given in Appendix S1.
(Co-Editor: Qi Xie)
This work was supported by the National Key Basic Research Program of the People's Republic of China (2012CB944801 and 2011CB915402), and the National Science Foundation of China (30970270 to J.J.L.).