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. 2006 Dec;18(12):3491–3501. doi: 10.1105/tpc.106.047266

The Female Gametophyte and the Endosperm Control Cell Proliferation and Differentiation of the Seed Coat in Arabidopsis[W]

Mathieu Ingouff 1, Pauline E Jullien 1, Frédéric Berger 1,1
PMCID: PMC1785409  PMID: 17172356

Abstract

Double fertilization of the female gametophyte produces the endosperm and the embryo enclosed in the maternal seed coat. Proper seed communication necessitates exchanges of signals between the zygotic and maternal components of the seed. However, the nature of these interactions remains largely unknown. We show that double fertilization of the Arabidopsis thaliana female gametophyte rapidly triggers sustained cell proliferation in the seed coat. Cell proliferation and differentiation of the seed coat occur in autonomous seeds produced in the absence of fertilization of the multicopy suppressor of ira1 (msi1) mutant. As msi1 autonomous seeds mostly contain autonomous endosperm, our results indicate that the developing endosperm is sufficient to enhance cell proliferation and differentiation in the seed coat. We analyze the effect of autonomous proliferation in the retinoblastoma-related1 (rbr1) female gametophyte on seed coat development. In contrast with msi1, supernumerary nuclei in rbr1 female gametophytes originate mainly from the endosperm precursor lineage but do not express an endosperm fate marker. In addition, defects of the rbr1 female gametophyte also reduce cell proliferation in the ovule integuments before fertilization and prevent further differentiation of the seed coat. Our data suggest that coordinated development of the seed components relies on interactions before fertilization between the female gametophyte and the surrounding maternal ovule integuments and after fertilization between the endosperm and the seed coat.

INTRODUCTION

Organs consist of multiple neighbor tissues containing cells with specific proliferating and differentiating patterns. Precise spatial and temporal regulatory mechanisms must take place to integrate growth (cell division and cell elongation), patterning, and differentiation between the multiple tissues to enable proper organ development.

In flowering plants, a striking example of such complex coordinated control of growth and differentiation occurs during the reproductive phase. In Arabidopsis thaliana, sexual reproduction takes place in the haploid female gametophyte embedded within the diploid integuments of the ovule. The female gametophyte (FG) in Arabidopsis originates from the surviving haploid meiotic megaspore (stage FG1), which undergoes three successive mitotic divisions (stages FG2, FG3, and FG4) generating a syncytium with eight nuclei (Yadegari and Drews, 2004). At stages FG5 and FG6, cellularization and differentiation take place to generate a seven-cell structure consisting of three antipodals, two synergids, and the two female gametes, the egg cell and the central cell, with one homodiploid nucleus resulting from the fusion of the two haploid polar nuclei (Yadegari and Drews, 2004). After degeneration of the three antipodals (stage FG7), the mature female gametophyte contains the female gametes and the synergids and becomes competent for fertilization (Yadegari and Drews, 2004). Double fertilization consists of the fusion between one sperm cell and the egg cell, resulting in the diploid embryo, and between the second sperm cell and the homodiploid central cell, leading to the development of the triploid endosperm.

In response to fertilization, ovule integuments in Arabidopsis differentiate into the seed coat that protects the endosperm and the embryo. Accumulation of tannins (proanthocyanidins [PAs]) is initiated after fertilization in the endothelium of the seed coat (Debeaujon et al., 2003). PA synthesis is controlled by TRANSPARENT TESTA GLABRA1 (TTG1) (Debeaujon et al., 2000) and TTG2 (Johnson et al., 2002). Loss of function in TTG2 affects the capacity of seed coat cells to elongate and, in turn, reduces endosperm growth and seed size (Garcia et al., 2005). Hence, the maternal diploid seed coat regulates endosperm growth, which consequently affects the final seed size.

Multiple sources of evidence also demonstrate the role played by the endosperm in the control of cell elongation in the seed coat. The major phase of seed growth parallels the initial endosperm growth characterized by rapid successive rounds of mitoses devoid of cytokinesis (syncytial development) (Garcia et al., 2005). Specific production of diphtheria toxin A in the developing endosperm caused a rapid arrest of seed growth (Weijers et al., 2003). Genetic analyses of the HAIKU pathway revealed that reduced syncytial endosperm growth prevents cell elongation in the seed coat, leading to smaller seeds (Garcia et al., 2003, 2005; Luo et al., 2005). The link between endosperm growth and seed coat cell elongation is also supported by parthenogenetic seed development in the fertilization-independent seed (fis) mutants. This mutant class consists of medea, fis2, fertilization independent endosperm (fie), and multicopy suppressor of ira1 (msi1) (Chaudhury et al., 1997; Ohad et al., 1999; Köhler et al., 2003; Guitton et al., 2004). In the absence of fertilization, fis-class mutants develop autonomous seeds characterized by an autonomous endosperm developing from the central cell and growth of the seed coat. The parthenogenetic embryo developing in msi1 autonomous seeds aborts rapidly and thus is unlikely to contribute to cell elongation in the seed coat. MSI1 and other FIS genes encode the four members of the conserved core of the class 2 Polycomb group complex (Grossniklaus et al., 1998; Ohad et al., 1999; Luo et al., 2000; Köhler et al., 2003; Guitton et al., 2004). It was shown that FIE and MIS1 interact with the RETINOBLASTOMA-RELATED homolog RBR1 in Arabidopsis (Mosquna et al., 2004) and other plants (Ach et al., 1997). Supporting this interaction, spontaneous nuclear proliferation has been observed in female gametophytes in the absence of fertilization of the rbr1 mutant in Arabidopsis (Ebel et al., 2004). Thus, it has been proposed that RBR1 interacts with the FIS pathway in the control of proliferation of the female gametophyte.

Although cell elongation in the seed coat driven by growth in the endosperm appears to play a major role in the control of seed size, cell proliferation occurs in the seed coat after fertilization (Garcia et al., 2005) and may also play a role in seed size control. Increased cell proliferation in the seed coat causes increased endosperm growth and seed size (Canales et al., 2002; Schruff et al., 2006). However, it is not known whether cell proliferation is regulated in the seed coat in response to fertilization. Besides interactions involving cell proliferation and elongation in the seed coat, evidence has been obtained for interaction between the endosperm and seed coat differentiation in barley (Hordeum vulgare) and maize (Zea mays) (reviewed in Chaudhury and Berger, 2001), but such evidence is lacking in Arabidopsis. One of the steps of integument differentiation in Arabidopsis involves the synthesis of flavonoids and is triggered by fertilization (Debeaujon et al., 2003), but the source of the signal involved remains unknown.

Here, we show that rapidly after fertilization, the endosperm triggers sustained cell division in the seed coat. Such active proliferation is also mimicked in the seed coat of autonomous seeds in msi1 but, surprisingly, not in rbr1. Seed coat differentiation is also initiated in msi1 autonomous seeds but is prevented in the rbr1 mutant. Further analyses demonstrate that loss of function of RBR1 alters female gametophyte development and does not generate autonomous seeds. We further observe that defects in the rbr1 female gametophyte affect cell proliferation in the ovule integuments before fertilization, suggesting a control of the female gametophyte during cell proliferation in the ovule integuments.

RESULTS

Autonomous Development in Emasculated msi1 and rbr1 Mutants

To determine further the relative roles played by fertilization signals, the embryo, and the endosperm on seed coat development, we studied autonomously developing seed-like structures produced by the msi1 and rbr1 mutants in the absence of fertilization. In the absence of fertilization, the wild-type ovule integuments showed limited growth (Figures 1A and 1B). By contrast, autonomous seed development induced by msi1 was accompanied by marked integument growth that mimicked to some extent seed coat development in wild-type fertilized seeds (Figures 1C to 1F). Loss of function of MSI1 has a recessive effect (Köhler et al., 2003) and therefore should not directly affect msi1/+ integuments. Seed-like structures produced by msi1 female gametophytes contain only autonomous endosperm during the first days, when integument growth is observed. We thus hypothesized that msi1 autonomous endosperm development sustains ovule integument growth, likely reflecting a general role of the endosperm in this process in fertilized seeds.

Figure 1.

Figure 1.

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Confocal Sections of msi1 and rbr1 Mutant Autonomous Seeds.

Mature flower buds from the wild type ([A] and [B]) and msi1 ([E] and [F]) and rbr1 ([G] and [H]) mutants were emasculated and stained with Feulgen at 2 d ([A], [C], and [E]) or 3 d ([B], [D], and [F]) after emasculation. Emasculated mature flower buds from the wild type were pollinated and stained with Feulgen at 2 d (C) or 3 d (D) after pollination. In absence of fertilization, the msi1 mutant forms an autonomous seed that develops a proliferating autonomous endosperm accompanied by seed coat growth ([E] and [F]; compare with the wild type in [A] and [B], respectively). (E) represents an example of autonomous endosperm division first observed in a small fraction of the population of ovules. In the rbr1 mutant ([G] and [H]), overproliferation of nuclei occurs but is not paralleled by integument growth. cc, central cell; ec, egg cell; emb, embryo; end, endosperm; sn, supernumerary nuclei; syn, synergid. Bars = 25 μm.

Overproliferation of nuclei was also observed at the micropylar pole, where the egg cell and synergids differentiate (Ebel et al., 2004). However, we could not find evidence for supernumerary division of the egg or the synergids, although in-depth analysis of egg cell–specific and synergid-specific fate marker expression should be required before a conclusion is drawn. Nuclear proliferation has been reported in rbr1 female gametophytes, which could lead to the production of seed-like structures containing autonomous endosperm (Ebel et al., 2004). We tested whether loss of function in RBR1 in the haploid female gametophyte affects integument development several days after emasculation (DAE). Although nuclear proliferation in the rbr1 female gametophyte could produce up to 20 nuclei by 3 DAE (Figure 1H), the size of the rbr1 seed-like structures was similar to that of wild-type ovules (cf. Figures 1G and 1H with 1A and 1B, respectively). The lack of response from the integuments could originate from a semidominant action of rbr1 on ovule integuments, which would make the integuments incompetent to respond to endosperm. This is unlikely, as rbr1/+ plants produce wild-type-size seeds from the segregating wild-type gametophytes. Alternatively, the proliferating structure derived from unfertilized rbr1 gametophytes may not achieve an endosperm identity enabling integument growth. We further analyzed in detail the effect of fertilization and autonomous development in msi1 and rbr1 on seed coat development and in parallel analyzed the developmental identity of autonomous proliferating rbr1 gametophytes.

Analysis of Integument Cell Proliferation

Seed coat growth results from the combination of cell proliferation and cell elongation. The endosperm contributes significantly to seed coat cell elongation at 4 d after pollination (Garcia et al., 2003). However, it was not clear whether endosperm growth could influence cell proliferation in the seed coat earlier than 4 d after pollination. To estimate the effect of fertilization on cell proliferation activity in the seed coat, we quantified the total number of cells per seed, which underwent the transition from the G2-phase to mitosis using the reporter construct PCyclinB1;2:glucuronidase (PCycB1;2:GUS) (Schnittger et al., 2002). This estimated mitotic activity was compared between the developing seed coat and the integuments of nonfertilized ovules (Figure 2). In the absence of fertilization, ovule integuments showed continuous mitotic activity for 4 DAE with an initial increase around ovule maturation and a sharp decline after 2 DAE. A similar trend was observed in the seed coat of developing seeds, with cell proliferation arrest corresponding to the rapid transition to cell elongation in the seed coat at ∼4 d after pollination. However, cell proliferation in developing seeds was more intense and sustained compared with that in nonfertilized ovules, suggesting that signals produced by fertilization, the endosperm, and/or the embryo promoted cell proliferation in the seed coat.

Figure 2.

Figure 2.

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Time-Course Evaluation of Mitotic Activity in the Integument Cells of msi1 and rbr1 Autonomous Seeds.

The number of cells expressing the PCycB1;2:GUS mitotic marker in the integument cells was estimated from 0 to 5 d after pollination (DAP) for the wild type (dark gray bars) (0 d after pollination corresponds to emasculation time, 12 h before pollination). In parallel, the same experiment was performed on emasculated pistils from 0 to 5 d after emasculation on the wild type (light gray bars), msi1/+; PCycB1;2:GUS/+ (white bars), and rbr1/+; PCycB1;2:GUS/+ (black bars). n represents the number of ovules/seeds from which integument cells expressing GUS were counted. Error bars represent sd.

To identify the origins of signals stimulating cell proliferation in the seed integuments after fertilization, we introgressed the cell division marker PCycB1;2:GUS into msi1 and rbr1 mutants and estimated the cell proliferation rate of integuments in the absence of fertilization. We first analyzed msi1-2/+; PCycB1;2:GUS/PCycB1;2:GUS plants. From 3 DAE, two groups of ovules could be distinguished according to their mitotic activity. One of the groups showed higher activity than the wild type and therefore likely resulted from the msi1 mutation. These ovules showed increased size, as expected for msi1 autonomous seeds by autonomous endosperm development after 3 DAE. The 1:1 ratio between the two groups indicated a gametophytic genetic segregation of msi1 (85 wild-type:71 msi1 ovules analyzed; χ2 test, P > 0.26). Autonomous seeds produced by ovules carrying msi1 alleles could be distinguished from wild-type unfertilized ovules by increased mitotic activity in the integuments (Figure 2). Compared with wild-type emasculated ovules, seed coat cells of msi1 autonomous seeds showed a distinctively prolonged phase of cell proliferation. This was parallel to the onset of autonomous proliferation in the endosperm at 1.5 DAE (data not shown). The first division of the parthenogenetic zygote from msi1 ovules is initiated after 4 DAE (Guitton and Berger, 2005) and thus is unlikely to contribute to the increased mitotic activity in the msi1 autonomous seed coat, which is observed before 4 DAE. We concluded that autonomous endosperm growth is responsible for the increased mitotic activity in the integuments in msi1 autonomous seeds. As a similar degree of cell proliferation is eventually achieved in the msi1 autonomous seed coat and in fertilized seeds, we propose that cell proliferation in the developing seed coat is enhanced by a signal originating from the developing endosperm after fertilization.

In contrast with msi1, we did not observe any increased mitotic activity in the integuments of rbr1 emasculated ovules at 3 DAE. The initial integument cell proliferation in maturing rbr1 ovules was comparable to that in the wild type (Figure 2). However, rbr1/+ plants produced two classes of ovules with either wild-type or reduced mitotic activity that were distinguishable as early as 1 DAE. The relative proportion of each class was compatible with the expected 1:1 segregation ratio for wild-type versus rbr1 ovules (71 wild-type ovules with cell proliferation and 59 rbr1 ovules with reduced proliferation; χ2 test, P > 0.29). Although all ovules with a wild-type female gametophyte showed mitotic activity comparable to that in the wild type, most seed-like structures with reduced mitotic activity displayed abnormal female gametophyte development resulting from RBR1 loss of function. In the rbr1 mutant, the mitotic activity remained unchanged until 3 DAE (Figure 2). We thus proposed that rbr1 effects on female gametophyte development limit cell proliferation in the integuments and that supernumerary nuclear divisions in the rbr1 female gametophyte in the absence of fertilization are not sufficient to trigger a sustained cell proliferation in ovule integuments, as observed in msi1 autonomous seeds.

Analysis of Seed Coat Differentiation

We further analyzed the differentiation of integument cells in the absence of fertilization in msi1/+ and rbr1/+ mutants. Fertilization triggers the accumulation of PA pigments in the endothelium cell layer of the seed coat, which can be detected as a red stain by vanillin assay (Debeaujon et al., 2003) (Figure 3B). Wild-type emasculated ovules showed no notable staining at 2 DAE (Figure 3A). By contrast, msi1 autonomous seeds accumulated PA in the seed coat (n = 324) (Figure 3C). Time-course analysis of PA synthesis during autonomous seed development in msi1 mutants further showed that PA accumulation in msi1 autonomous seeds closely paralleled the postfertilization differentiation of seed coat cells in wild-type seeds (see Supplemental Figure 1 online). These results suggest that PA accumulation in the endothelium is induced by autonomous msi1 and wild-type endosperm. However, msi1 autonomous seeds also contain a parthenogenetic embryo (Guitton and Berger, 2005). Unlike msi1 autonomous seeds, autonomous seeds from fis2 solely develop an autonomous endosperm (Guitton et al., 2004). We observed that PA accumulation also occurs in fis2 autonomous seeds (Figure 3D).

Figure 3.

Figure 3.

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PA Synthesis in msi1, fis2, and rbr1 Autonomous Seeds.

PAs are specifically deposited in the endothelium cells of the seed coat upon fertilization. Whole-mount vanillin staining enables direct visualization of PA by staining PA-accumulating cells in red.

(A) Absence of PA in integuments of a wild-type unfertilized ovule.

(B) Uniform detection of PA in the endothelium of a wild-type seed at 3 d after pollination.

(C) Uniform detection of PA in the endothelium of an msi1 autonomous seed at 3 DAE.

(D) Accumulation of PA in the endothelium of a fis2 autonomous seed at 2 DAE.

(E) Absence of PA in integument cells of an rbr1 autonomous seed at 2 DAE.

Arrowheads indicate supernumerary nuclei typical of the rbr1 mutant. C, chalazal pole; cc, central cell; end, endosperm; M, micropylar pole. Bars = 25 μm.

In conclusion, loss of function of MSI1 triggers the development of autonomous endosperm, which is sufficient to induce increased mitotic activity, PA synthesis, and cell elongation in the seed coat, although fertilization signals and embryo development are absent. We thus propose that endosperm development in wild-type seeds is sufficient for proper seed coat growth and differentiation.

In contrast with msi1, rbr1/+ integuments surrounding rbr1 female gametophytes with nuclear proliferation remained unstained after a vanillin assay, as in wild-type emasculated ovules (n = 422) (Figure 3E). Excessive nuclear proliferation in the rbr1 female gametophyte does not trigger the growth and differentiation of a seed coat. Hence, the rbr1 female gametophyte likely produces a tissue that does not have a proper endosperm identity. Alternatively, the rbr1 female gametophyte may not reach complete maturity and may retain some proliferative capacity typical of development stages earlier than stage FG5, when cellularization is initiated and fusion of the polar nuclei forms the central cell (Yadegari and Drews, 2004).

Developmental Identity of the rbr1 Proliferating Gametophyte

To determine cell fate in syncytial proliferating rbr1 female gametophytes, we analyzed the expression of the endosperm-specific marker KS117 in the rbr1/+ genetic background. KS117 is not detected in the wild-type female gametophyte but is expressed after fertilization in the endosperm as early as stage IV/V (8/16 nuclei) (Ingouff et al., 2005). KS117 is expressed during msi1 autonomous seed development (Guitton and Berger, 2005). At 3 DAE, supernumerary nuclei in rbr1 female gametophytes did not exhibit any expression of KS117 (n = 252). This absence of endosperm marker expression showed that the rbr1 female gametophyte does not develop an endosperm-like structure, suggesting that overproliferating tissues in rbr1 ovules retain a gametophytic identity in the absence of fertilization.

We analyzed the expression of PMYB98:green fluorescent protein (PMYB98:GFP), a marker of female gametophyte maturation (Kasahara et al., 2005), during rbr1 female gametophyte development. At stage FG5, when the migration of polar nuclei occurs to form the central cell (Drews and Yadegari, 2002), PMYB98:GFP was expressed uniformly in the wild-type female gametophyte except in the antipodals at the chalazal pole (Figure 4A) (Kasahara et al., 2005). At maturity (stage FG7), PMYB98:GFP expression became restricted mainly to the two synergids, but a weak expression remained in the egg cell and the central cell (Figure 4B). After fertilization, PMYB98:GFP persisted in the intact synergid and was detected in the endosperm (Figure 4C). In rbr1 female gametophytes, PMYB98:GFP was expressed at very high levels in synergids, suggesting that synergid identity was not perturbed by rbr1. However, PMYB98:GFP expression was never restricted to the synergids, as in wild-type mature female gametophytes, and it persisted in the chalazal part of the female gametophyte showing nuclear overproliferation (Figure 4D). As rbr1 female gametophyte proliferating tissue did not assume an endosperm identity, the unrestricted MYB98 expression suggests that an abnormal gametophytic identity persists in overproliferating rbr1 female gametophytes. Supernumerary nuclei in the proliferating rbr1 gametophytic tissue expressed PMEDEA:GUS (Figure 5C), a marker of the central cell lineage detected from stage FG5 specifically in polar nuclei (Figure 5A) and after the fusion of polar nuclei in the central cell (stage FG6) (Figure 5B) (Luo et al., 2000). This finding suggested that supernumerary nuclei in rbr1 female gametophytes derive mainly from unrestricted proliferation of the central cell lineage. This result was supported by the absence of obvious proliferation of cells from the egg apparatus, which contained two synergids and a single egg cell, as in wild-type female gametophytes (see Supplemental Figure 2 online). We further analyzed the mitotic activity of the various cells of the rbr1 female gametophyte in rbr1/+; PCycB1;2:GUS/+ plants. After emasculation, we could not detect any GUS activity in ovules from wild-type pistils (Figure 6A; n = 435). By contrast, GUS staining was observed at low frequency in a low percentage of rbr1 female gametophytes at 3 DAE (five positive ovules; n = 816). In all of these cases, GUS activity was detected only around nuclei derived from the central cell and not from the egg apparatus (Figure 6B; n = 5). This shows that extra nuclei in rbr1 female gametophyte derive mostly from the proliferation of the central cell lineage.

Figure 4.

Figure 4.

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Expression of the PMYB98:GFP Marker in a Female Gametophyte of the rbr1 Mutant.

(A) to (C) Wild type. Expression pattern of the PMYB98:GFP marker in the female gametophyte.

(A) PMYB98:GFP is detected in all parts of the female gametophyte before fusion of the polar nuclei (stage FG5), except in antipodals.

(B) Later, GFP is restricted mainly to the synergids, but a weak signal remains in the egg cell and the central cell (stage FG7).

(C) After fertilization, expression is detected in the intact synergid and in the endosperm at the four-nuclei stage.

(D) rbr1 mutant. Expression is never restricted to the synergids and remains in all parts of the rbr1 female gametophyte. The highest signal intensity is still detected in the synergids, as in the wild type.

ap, antipodals; cc, central cell; dsyn, degenerate synergid; ec, egg cell; end, endosperm; pn, polar nuclei; sn, supernumerary nuclei; syn, synergid; zy, zygote. Bars = 10 μm for (A) and 25 μm for (B) to (D).

Figure 5.

Figure 5.

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Expression of the PMEDEA:GUS Marker in a Female Gametophyte of the rbr1 Mutant.

(A) and (B) Wild type. PMEDEA:GUS staining in the female gametophyte was described previously by Luo et al. (2000). GUS activity is detected in the polar nuclei (A) and in the central cell upon fusion of the polar nuclei (B).

(C) rbr1 mutant. GUS staining is detected in supernumerary nuclei (arrowheads) in the rbr1 female gametophyte.

cc, central cell; ea, egg apparatus; pn, polar nuclei. Bars = 10 μm.

Figure 6.

Figure 6.

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Expression of the Mitotic Marker PCycB1;2:GUS in a Female Gametophyte of the rbr1 Mutant.

(A) Wild type. Salt-and-pepper GUS staining pattern in a cleared ovule.

(B) rbr1 mutant. Marked GUS expression in the supernumerary nuclei in the chalazal and central regions of the female gametophyte. No GUS activity is detected in the egg apparatus.

ea, egg apparatus; fg, female gametophyte; oi, ovule integuments. Bars = 10 μm.

To further understand the origin of the defects of rbr1 female gametophytes, we established the expression pattern of RBR1 during female gametophyte development. The whole RBR1 locus containing the promoter and the downstream sequences was fused in frame with the monomeric Red Fluorescent Protein1 (mRFP1) (Campbell et al., 2002). The PRBR1:RBR1-mRFP1 translational fusion construct was transformed into selected rbr1-2 mutant plants. Expression of the RBR1-mRFP1 fusion rescued the rbr1-2 mutation, hence demonstrating functionality (see Supplemental Figure 3 online). The dynamic localization of RBR1-mRFP1 was then studied by confocal microscopy during female gametophyte development (Figure 7). The fusion protein RBR1-mRFP1 showed a salt-and-pepper distribution in the ovule integument cells, as observed previously for RBR1 mRNAs in root meristems (Wildwater et al., 2005). This probably reflects the control of RBR1 expression by the cell cycle machinery. In the female gametophyte, the RBR1-mRFP1 fusion protein was detected mostly in nuclei (Figure 7). At stage FG1, RBR1-mRFP1 was present in the nucleus of the surviving megaspore mother cell (Figure 7A). RBR1-mRFP1 expression persisted in all nuclei during the three synchronous mitoses leading to stages FG2/3 (Figure 7B), FG4 (Figure 7C), and FG5 (Figure 7D). During stage FG5, the overall expression of RBR1-mRFP1 decreased, and after polar nuclei fusion (stage FG6), RBR1-mRFP1 persisted mostly in the central cell of the female gametophyte (Figure 7E). At maturity, RBR1-mRFP1 was completely excluded from the egg apparatus and was expressed only in the central cell (stage FG7; Figure 7F). The restricted expression of RBR1 to the central cell at ovule maturity supports the notion that cell cycle arrest in this cell depends directly on RBR1 function. In conclusion, we propose that rbr1 female gametophytes proceed normally through the three successive mitoses (stages FG1 to FG4) but fail to arrest nuclear division after the third mitosis, when cellularization occurs and differentiation is initiated (Christensen et al., 1997). Our cellular and marker gene expression analyses indicate that the rbr1 female gametophyte differentiation is initiated but might not be complete.

Figure 7.

Figure 7.

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Dynamic Analysis of the PRBR1:RBR1-mRFP1 Fusion Protein during Female Gametophyte Development.

Confocal images from the RBR1-mRFP1 fusion protein under the control of its native promoter were obtained from 11 independent stable transformed plants.

(A) Ovule with the megaspore (arrowhead).

(B) Two-nucleate-stage ovule.

(C) Four-nucleate-stage ovule.

(D) Eight-nucleate-stage ovule.

(E) Maturing female gametophyte.

(F) Mature female gametophyte.

ap, antipodals; cc, central cell; ec, egg cell; nu, nucellus; oi, ovule integuments; syn, synergid. Bars = 5 μm for (A) and 10 μm for (B) to (F).

DISCUSSION

Fertilization Enhances Cell Proliferation and Triggers Differentiation of the Seed Coat

Our study on cell proliferation in the seed coat shows that a sustained mitotic activity is triggered after fertilization and persists until 3 d after pollination. In response to fertilization, the differentiation of ovule integuments into seed integuments is activated (Haughn and Chaudhury, 2005) with the accumulation of PAs in the endothelium (Debeaujon et al., 2003) and cell elongation (Garcia et al., 2005).

We showed that msi1 autonomous endosperm development triggers cell proliferation, cell elongation, and differentiation of the seed coat. Although a nonviable parthenogenetic embryo also develops in msi1 autonomous seeds, msi1 autonomous seed development results mainly from autonomous endosperm growth (Guitton and Berger, 2005). Autonomous seed development in fis2 involves only autonomous endosperm development (Guitton et al., 2004), which appears to be sufficient to trigger PA synthesis. No significant growth of the seed coat is observed in seeds in which only the egg cell is fertilized as a result of the production of a single sperm cell in A-type cyclin-dependent kinase1 mutant pollen (Nowack et al., 2006). Similarly, ablation of endosperm in developing seeds results in extremely reduced seed coat development (Weijers et al., 2003). Thus, we propose that fertilization of the egg cell and consequent embryo development may not play a major role in seed coat growth. We propose that activation of the central cell and consequent endosperm development are probably the key players in the transition from ovule integument proliferation to seed coat growth and differentiation in the wild-type seed. The signals originating from the maturing female gametophyte and developing endosperm, involved in ovule integument and the seed coat, respectively, remain to be identified.

The rbr1 Mutation Does Not Trigger Autonomous Seed Development

In absence of fertilization, fis-class mutants, including mea, fis2, fie, and msi1, develop an autonomous endosperm from the unfertilized central cell accompanied by autonomous seed development and parthenocarpy (Köhler et al., 2003; Guitton et al., 2004). This common phenotype is supported by the ability of the three FIS Polycomb group proteins together with MSI1 to assemble in a complex (Köhler et al., 2003; Bracha-Drori et al., 2004; Chanvivattana et al., 2004).

MSI1 and RBR1 homologs are able to bind to each other (Ach et al., 1997) and may form complexes with the Polycomb group partner FIE (Mosquna et al., 2004). Based on the biochemical data and the presence of overproliferating nuclei in rbr1 female gametophytes, it was reasonably proposed that this mutant phenotype was reminiscent of an autonomously developing endosperm typical of the fis Polycomb group mutant (Ebel et al., 2004). However, our study indicates that rbr1 ovules are unable to express an early endosperm marker and to induce endosperm-derived signals necessary for the differentiation of seed coat cells. Rather, although sustained nuclear proliferation is observed in the female gametophyte, rbr1 ovules degenerate in the absence of fertilization similarly to the wild type.

In conclusion, overproliferation of nuclei from rbr1 female gametophytes does not reflect autonomous endosperm development, and the rbr1 mutant is unable to promote an autonomous seed developmental program.

During the three successive mitoses of female gametophyte development (stages FG1 to FG5), RBR1 is expressed in all nuclei of the female gametophyte to become restricted to the female gametes (stage FG6). Eventually, RBR1 becomes confined to the central cell and excluded from the egg cell in the mature female gametophyte (stage FG7). This suggests that cell cycle arrest is different in the two female gametes, the central cell and the egg cell. Retinoblastoma proteins in mammals and plants are central cell cycle regulators that prevent cell division by blocking the transition from G1- to S-phase in the cell cycle (Inze, 2005). The restriction of RBR1 protein to the central cell in the mature female gametophyte suggests that this cell is arrested in G1-phase.

Confinement of RBR1 to the central cell further suggests that supernumerary nuclei in rbr1 mature female gametophytes originate from the central cell lineage and not from the egg apparatus lineage, as reported previously (Ebel et al., 2004). This is consistent with the expression of a marker of the central cell lineage in the chalazal pole of rbr1 female gametophytes and of the mitotic cyclin B1,2 reporter. Our microscopic analyses further suggest that the integrity of the egg apparatus is preserved in rbr1 ovules, which progress through the initial stages of gametophytic divisions. Excessive proliferation from the micropylar pole observed in previous studies (Ebel et al., 2004) may originate from a smaller fraction of rbr1 gametophytes affected by early arrest or by abnormal precocious cellular development. Additional specific markers for antipodals and the egg cell will be necessary to precisely establish cell fate identity in all classes of rbr1 female gametophytes.

The Female Gametophyte Controls Cell Proliferation in Ovule Integuments

Before fertilization, ovule growth implies coordinated growth between the enlarging female gametophyte and the surrounding inner and outer integuments. Our results show that integument cell proliferation intensity is sustained as ovules reach maturity (Figure 2). This marked mitotic activity upon terminal ovule maturation is absent in rbr1 ovule integuments (Figure 2). This finding suggests that the perturbed proliferation rate in rbr1/+ integument cells results from defects in haploid female gametophytes. Our analyses of markers of the female gametophyte in the rbr1 mutant background suggest that rbr1 female gametophytes are affected when differentiation is initiated by stage FG5. We propose that final steps of female gametophyte maturation promote integument cell proliferation to enable terminal growth of the female gametophyte before fertilization. This suggests that the maturing female gametophyte conveys cell proliferation–promoting signals to the surrounding ovule integuments. However, the importance of such signals is likely to be only moderate, as integuments develop to some extent in spl mutant ovules, which do not contain embryo sacs (Schiefthaler et al., 1999; Yang et al., 1999).

It is unknown whether signals originating from surrounding ovule integuments toward the female gametophyte also exist. However, mutations affecting the initiation and development of the ovule integuments often lead to an absence or an early block of female gametophyte development, suggesting that proper ontogenesis of the female gametophyte also involves signals from the surrounding inner integument (Reiser et al., 1995; Elliot et al., 1996; Klucher et al., 1996; Baker et al., 1997; Schneitz et al., 1997).

In conclusion, reciprocal controls likely take place between the female gametophyte and its surrounding integuments before and after fertilization to integrate the multiple parameters necessary to ensure the coordination of sporophytic and gametophyte programs and successful transmission of the offspring.

METHODS

Plant Strains and Growth Conditions

The Arabidopsis thaliana rbr1 mutant alleles (Columbia accession) used in this study were rbr1-1 (SALK_012270; SALK collection), rbr1-2 (SALK_002946; SALK collection), and rbr1-3 (GABI_170G02; GABI-Kat collection) (Ebel et al., 2004). The msi1 mutant alleles were msi1-1 (Columbia accession) (Köhler et al., 2003) and msi1-2 (C24 accession) (Guitton et al., 2004). The fis2 mutant allele (fem17; Columbia accession) was provided by Gary Drews (Christensen et al., 1997).

The enhancer trap mGFP5 line KS117 (C24 accession) was identified after a screen in Jim Haseloff's collection (Haseloff, 1999; Ingouff et al., 2005). The transgenic line containing the construct PMEDEA:GUS reporter line (C24 accession) was provided by Abed Chaudhury (Luo et al., 2000). The transgenic seeds (Columbia accession) for the construct PMYB98:GFP were a gift from Gary Drews (Kasahara et al., 2005). The marker line (Landsberg erecta accession) containing the construct PCycB1;2:GUS was given by Arp Schnittger (Schnittger et al., 2002).

After 2 d of stratification at 4°C in the dark, seeds were germinated and grown on soil. Plants were cultured in a growth chamber under short days (8 h of light at 20°C/16 h of dark at 16°C; 60 to 70% RH) until rosettes were formed. Seedlings were transferred to long days at 20°C (14-h day/10-h night) to induce flowering and grown until seeds were harvested.

Microscopy

GUS assay was performed as reported previously (Boisnard-Lorig et al., 2001). After an overnight incubation at 37°C, stained developing pistils were dissected and ovules or seeds were cleared with a derivative of Hoyer's medium and observed microscopically with differential interference contrast optics (DM6000 B; Leica). Images were acquired with a DXM1200F digital camera (Nikon) and processed with Metamorph (version 6.2; Universal Imaging).

Fluorescence was imaged using laser scanning confocal microscopy (Zeiss LSM-510) for GFP with selective settings for GFP detection (excitation, 488 nm; emission, band-pass 510 to 550 nm) and nonspecific settings for autofluorescence detection (excitation, 543 nm; emission, long-pass 560 nm) and for mRPF1 (excitation, 543 nm; emission, band-pass 560 to 615 nm).

Image acquisition on fixed tissues was performed on a Zeiss LSM-510 laser scanning confocal microscope using the 488-nm excitation line of an argon laser and a long-pass emission filter at 510 nm. Digital image processing was performed with Adobe Photoshop and Adobe Illustrator (Adobe Systems).

Time-Course Evaluation of Mitotic Activity in the Integuments

The total number of cells at the G2/M transition was counted per ovule integument and per seed coat using the marker of mitotic activity, PCycB1;2:GUS. Analyses of cell division activity were performed on segregating wild type and rbr1-3 and msi1-2 mutants homozygous for PCycB1;2:GUS. Flowers at 1.5 d before anthesis were emasculated each day for 5 d to estimate integument cell proliferation. In parallel, emasculated pistils from wild-type plants at 1.5 d before anthesis were pollinated 24 h later with pollen from homozygous PCycB1;2:GUS plants. Samples were collected and stained for GUS. Measurement of any integument cell with GUS expression was obtained for individual ovules taken at random. In populations of ovules from msi1-1/+ plants, before 2 DAE, all ovules showed similar mitotic activity. After 3 DAE, two groups of ovules became distinguishable. Ovules from one group had mitotic activity similar to emasculated wild-type ovules, whereas ovules from the other group showed higher mitotic activity and were presumably msi1-1 mutant ovules. The average value considered for the graph was the mean from the latter group. Similarly, ovules from emasculated rbr1-3/+ plants segregated into two groups after 1 DAE. For consideration in the graph, the group with lower activity than the wild type was chosen.

Analysis of the Phenotype Conferred by rbr1

Pistils from rbr1-2 and msi1-1 mutants were emasculated 1 d before anthesis and collected 2 or 3 d later. Pistils were cut at the tip, stained with Schiff's reagent (Sigma-Aldrich), and embedded in LR White (Sigma-Aldrich) according to Braselton et al. (1996). The mutant lines rbr1-2 and msi1-1 can only be propagated as heterozygotes and produce pistils that contain 50% wild-type ovules and 50% ovules that display the mutant phenotype.

The PMEDEA:GUS and PMYB98:GFP reporter gene constructs and the KS117 endosperm marker were introgressed respectively into rbr1-1 and rbr1-2 by crossing. Analyses were performed on rbr1 mutants homozygous for the marker. At 2 DAE, pistils from rbr1-2; KS117/KS117 were dissected to determine GFP activity. Emasculated pistils from rbr1-1; PMEDEA:GUS/PMEDEA:GUS and rbr1-1; PMYB98:GFP/PMYB98:GFP were analyzed at 1 DAE.

Whole-Mount Staining for Detection of PAs

Emasculated pistils at 1.5 d before anthesis from rbr1-2/+ and msi1-1/+ mutants were left unpollinated for 3 d to promote autonomous seed development. Emasculated pistils at 1.5 d before anthesis from the wild type were pollinated at 1.5 d after emasculation to be used as a reference.

For the time-course analysis of PA synthesis during autonomous seed development in msi1-1, 10 pistils at 1.5 d before anthesis were emasculated every day for 3 d.

Ovules and seeds were dissected from pistils and siliques, respectively, and incubated in an acidic solution (6 n HCl) of 1% (w/v) vanillin (Sigma-Aldrich) at room temperature (Aastrup et al., 1984). Vanillin reacts in the presence of PAs that specifically accumulate in the endothelium layer of the seed coat, generating a red product (Debeaujon et al., 2003). Microscopic observations were performed with differential interference contrast optics (DM6000 B; Leica). Images were acquired with a DXM1200F digital camera (Nikon) and processed with Metamorph (version 6.2; Universal Imaging).

RBR1:RBR1-mRFP1 Plasmid Construction and Transformation

A 6968-bp fragment of RBR1 containing 2 kb upstream of the ATG until the last codon before the termination codon of the gene was amplified by PCR with the KOD-plus-PCR kit (Toyobo) and cloned directionally between the Gateway attL recombination sites of the plasmid pENTR-D-TOPO (Invitrogen) to generate the pENTR-D-TOPO-PRBR1:RBR1 entry vector.

The fragment containing the cassette Gateway attR, mRFP1, and the nopaline synthase (NOS) terminator (K7GW-mRFP1-NOS) was cloned into the binary plasmid pALLIGATOR2 (Bensmihen et al., 2004; http://www.isv.cnrs-gif.fr/jg/alligator/vectors.html) to generate pAlli2-K7GW-mRFP1-NOS.

Recombination reactions (attL × attR) were performed between the destination vector pAlli2-K7GW-mRFP1-NOS and the pENTR-D-TOPO-PRBR1:RBR1 entry vector to produce the vector pAlli2-PRBR1:RBR1-mRFP1-NOS.

rbr1-2/+ mutant (kanamycin-selected) plants were transformed using the Agrobacterium tumefaciens–mediated floral dip method (Clough and Bent, 1998). The pALLIGATOR vectors contain the GFP gene driven by a seed storage protein promoter, At2S3, enabling direct visual selection. Primary seed transformants were collected based on their fluorescence level in the seed and further selected on Murashige and Skoog plates supplemented with kanamycin (50 mg/L). Eleven transgenic lines were obtained, all showing a similar pattern of expression. Complementation was achieved by transformation of rbr1-2/+ plants with the RBR1 locus fused to mRFP1. Complementation was analyzed on one line.

Accession Numbers

Accession numbers for the MSI1 and RBR1 genes are At5g58230 and At3g12280, respectively.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Time-Course Analysis of PA Synthesis during Autonomous Seed Development in the msi1 Mutant.

  • Supplemental Figure 2. Frontal View of the rbr1 Female Gametophyte.

  • Supplemental Figure 3. Complementation of the rbr1-2 Mutant with the RBR1 Locus Fused to mRFP1.

Supplementary Material

[Supplemental Data]
plntcell_tpc.106.047266_index.html (4.8KB, html)

Acknowledgments

We thank Lars Henning for providing the msi1-1 allele, Arp Schnittger for his gift of the marker PCycB1;2:GUS, Abed Chaudhury for the marker PMEDEA:GUS, and Gary Drews for the marker PMYB98:GFP. We thank the ABRC for providing us with the rbr1-1 and rbr1-2 mutant alleles and GABI-Kat for the rbr1-3 mutant allele. We are grateful to Francois Parcy for providing us with the Alligator plasmids. P.E.J. and F.B. were supported by the Temasek Lifesciences Laboratory and by the National University of Singapore. M.I. was supported by the Singapore Millennium Foundation.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Frédéric Berger (fred@tll.org.sg).

[W]

Online version contains Web-only data.

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Associated Data

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Supplementary Materials

[Supplemental Data]
plntcell_tpc.106.047266_index.html (4.8KB, html)
plntcell_tpc.106.047266_1.pdf (9.2MB, pdf)
plntcell_tpc.106.047266_2.pdf (3.4MB, pdf)
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