Isolation of synaptonemal complexes from lily microsporocytes

Plant Science, 86 (1992) 115-124

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Elsevier Scientific Publishers Ireland Ltd.

Isolation of synaptonemal complexes from lily microsporocytes T o s h i o O h y a m a a, Y u k i o I w a i k a w a b, T o s h i y u k i K o b a y a s h i c, Y a s u o H o t t a c a n d Satoshi T a b a t a c aDepartment of Biology, College of Education, Fukui University, Fukui 910, bDepartment of Biology, College of General Education, Nagoya University and CDepartment of Biology, School of Science, Nagoya University, Nagoya 464-01 (Japan) (Received November 5th, 1991; revision received June 5th, 1992; accepted June 9th, 1992)

Synaptonemal complexes (SCs) were isolated from microsporocytes of Lilium. Nuclei prepared from microsporocytes at pachytene in meiotic prophase were treated with DNase II and SCs released from the nuclei were purified by sedimentation through a density gradient of Nycodenz. The SC fraction thus obtained contained fibrous structures consisting of two parallel filaments as major components. They were - 10/xm in length and 200 nm apart from each other and were considered to be the segments of the lateral elements. According to sodium dodecyl sulfate (SDS)-gel electrophoretic analyses, twenty or more proteins were identified as components of the SC fraction. Among these, five proteins having M r values of 42, 50, 52 116 and 140 kDa were consistently present in various preparations. Antiserum raised against the isolated SCs recognized several kinds of antigens including the proteins of 42, 50 and 52 kDa, which were likely to be candidates for constituents of SCs.

Key words: meiosis; mJcrosporocytes; Lilium; synaptonemal complex

Introduction

Synaptonemal complexes (SCs) are tripartite structures consisting of two lateral elements and a central element, distinctively observed between paired homologous chromosomes during zygotene-pachytene in meiotic prophase. Genetic and cytological evidence have suggested that formation of SCs is intimately correlated with the meiotic recombination between homologous chromosomes [1-5]. Despite the importance of the possible roles in synapsis and homologous recombination in meiosis, SCs have not often been subjected to intensive biochemical analyses. Several investigators have attempted to isolate SCs from meiocytes of hamster spermatocytes [6], mouse spermatocytes Correspondence to: Dr. Toshio Ohyama, Department of Biology, College of Education, Fukui University, 9-1, Bunkyo 3-chome, Fukui-shi 910, Japan.

[7], rat spermatocytes [8] and oocytes of flour moth [9]. However, most of the SCs prepared were only useful for further qualitative analyses mainly due to contamination with nuclear matrices. Using rat spermatocytes, however, Heyting et al. [10] have isolated SCs, pure enough for antibody preparation by avoiding irreversible aggregation of nuclear matrices during preparation. Heyting and colleagues [11-13] have identified four nuclear proteins with Mr values of 30, 33, 125 and 190 kDa as components of SC. In addition, Smith and Benavente [14] has recently identified an SC protein having a Mr of 48 kDa in rat spermatocytes. Biochemical studies concerning the construction of the SC has proceeded using mammal spermatocytes, but there is little information about the biochemical nature of the SC in higher plants. In this paper, we report the improvement of our previous method [15] for isolation of SCs from lily microsporocytes, referring to the procedure of

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Heyting [10]. SCs isolated were then subjected to preliminary characterization of their morphology and protein composition with antiserum raised against isolated SCs. Materials and Methods

Isolation of nuclei Microsporocytes were obtained from anthers of lily, Lilium longiflorum Thunb. var Hinomoto. Estimation of the meiotic stage of the cells was based on the correlation between bud length and the meiotic stage [16] after cytological examination. Extruded microsporocytes were pooled and washed several times with White's medium to remove most of the contaminating tapetal nuclei, then stored at -20°C in White's medium containing 50% (v/v) glycerol as described elsewhere [17]. All the steps of nuclear isolation were performed at 4°C. A swinging bucket rotor was used in the centrifugation steps unless otherwise stated. Microsporocytes at pachytene were used as a source of nuclei. The nuclei were isolated by the method of Willmitzer and Wagner [18] with some modification. The microsporocytes were suspended in nuclear isolation medium (NIM) containing 50% (v/v) glycerol. NIM contains 10 mM Tris-HCl (pH 7.0), 10 mM KCI, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.15 mM spermine, 0.5 mM spermidine, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM 3-aminobenzamidine, 0.25 M sucrose and 10% (v/v) glycerol. The cells were disrupted by nitrogen decompression with the aid of a Parr Cell Disruption Bomb (PARR instrument company, USA) at a pressure of 1200 lb/in.2. The homogenate derived from 10 ml of the cell suspension was diluted with an equal volume of NIM and was passed through a layer of nylon mesh (opening 100 /zm) to remove unbroken tissues. The filtrate was centrifuged at 350 x g for 10 min. After sedimentation, the nuclear pellet was resuspended in 20 ml of NIM supplemented with 0.1% (v/v) Nonidet P-40 and centrifuged at 350 × g for 10 min.. This washing step was repeated twice. The pellet was gently suspended in 20 ml of NIM containing 35% (v/v) Percoll (Pharmacia, Sweden) and the suspension was centrifuged at 600 × g for 20 min.. The crude nuclear

pellet was resuspended in a small volume of NIM and the suspension was loaded onto a Percoll gradient which had been formed by centrifuging 20 ml of 35% Percoll in NIM in a 50-ml tube at 48 000 x g for 1 h with a fixed angle rotor. The gradient was centrifuged at 4000 x g for 30 min. and the nuclei banded near the bottom were collected with a Pasteur pipette. The nuclear fraction was diluted with NIM and sedimented by centrifugation at 350 x g for 10 min. This process was repeated three times to obtain the purified nuclei used as a source of synaptonemal complexes.

Isolation of SCs Isolation of SCs were carried out according to the method of Heyting et al. [10,11] with some modifications. All the centrifugation steps in purification of SCs were carried out with a swinging-bucket rotor at 4°C. Approximately 107 of the isolated nuclei were gently suspended in 5 ml of TED (containing 10 mM T r i s - H C l (pH 7.0), 0.1 mM EDTA, 0.5 mM DTT, 0.1% (v/v) Nonidet p-40, 0.2 mM PMSF and 0.1 mM 3aminobenzamidine). DNase II (type VIII, Sigma Chemical Co.) was added to the nuclear suspension at a final concentration of 200 Kunitz units/ml and the mixture was incubated at 20°C for 2 h with gentle shaking. After the DNase II treatment, the nuclei were sedimented by centrifugation at 4000 x g for 15 min, then washed twice with 10 ml of TED buffer. The pellet was resuspended in 1 ml of TED and the suspension was loaded onto 10 ml of a 20-40"/,, (w/v) linear gradient of Nycodenz (NYCOMED AS, Norway) in TED containing 20%(v/v) glycerol. After centrifugation at 26000 x g f o r 2 h , 0.2 ml aliquots were collected from the top of gradient. The fractions containing SCs, the middle part of the gradient as judged by light and electron microscopy, were combined and diluted with five volumes of the TED buffer and centrifuged at 26 000 x g for 20 min. The pellet was washed twice with 10 ml of the same buffer. Nucleolar fractions in the Nycodenz gradient were also collected and washed several times with TED buffer containing 0.5 M NaCI for gel analysis.

Light and electron microscopy Isolated nuclei and purified SCs were stained

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with silver reagent for observation as described elsewhere [19]. For cytological localization of the DNA in isolated SCs, the silver-stained specimens were stained with 4,6-diamino-2-phenylindol (DAPI) according to Kuroiwa et al. [20] and were observed by fluorescence under a microscope equipped with an epi-illuminator (BH2RKF, OLYMPUS, Japan). For electron microscopic observation of isolated SCs, carbon-collodion-coated grids were floated on the surface of a drop of the SC suspension for 1 min and the grids were placed in 2% (w/v) uranyl acetate for 10 min. for staining. After rinsing several times with distilled water, the grids were air-dried and examined in a JEM T-8 electron microscope.

Preparation of antiserum against SCs and immunocytochemical analysis The isolated SCs ( - 1 0 0 t~g of protein) were mixed with complete Freund's adjuvant and the mixture was injected under the skin of a BALB/C mouse. Booster injections were given twice at the 2nd and 4th week after the primary injection. The animal was bled on the 5th day after the final boost and the serum was collected after clotting. Control serum was obtained from a mouse which had not been immunized. Vectastain ABC kits (Vector Laboratories Inc., USA) were used for immunostaining of SCs. Unless otherwise noted, all incubations were carried out at room temperature in a humid chamber. Isolated pachytene nuclei were fixed in 2%(w/v) paraformaldehyde buffered with 5 mM borate at pH 8.0 for 10 min at 4°C. An aliquot of the fixed nuclear suspension was spread on a surface of a glass slide pretreated with poly-L-lysine. After rinsing twice in distilled water and once in 0.1 M K + phosphate (pH 7.0) containing 0.85% (w/v) NaC1, phosphate buffered saline (PBS), for 5 min each, the specimens were incubated in PBS containing 1.5% (v/v) normal horse serum for 30 min. After blotting the excess solution, anti-SC serum or the control serum at a dilution of 1:1000 in PBS was added to the specimens. The specimens were then treated with biotinylated anti-mouse IgG followed by horseradish peroxidase-conjugated ABC (avidin-biotin complex) reagents [21] according to

the manufacturer's instructions. Finally the specimens were incubated for 8 min. in 50 mM Tris-HC1 (pH 7.2) containing 0.5 mg/ml DAB (3,3'-diaminobenzidine) and 0.01% (v/v) H202. After washing twice in distilled water, the specimens were mounted in Euparal (Chroma gesellschft, Germany) and observed under a light microscope.

Isolation of nuclear proteins Total nuclear proteins were extracted as follows. The nuclei were lysed with a solution containing 6 M urea, 5 M NaC1 and 10 mM sodium phosphate (pH 7.3) and homogenized by sonication. After centrifugation at 10 000 × g for 20 min, 100% (w/v) trichloroacetic acid (TCA) was added to the supernatant fraction to a final concentration of 15% (w/v) and the mixture was chilled on ice for 30 min. The precipitate was collected by centrifugation at 10 000 x g for 10 min. The pellet was washed several times with acetone, then air-dried.

Gel electrophoresis and immunoblotting Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli [22], with 8-16% linear gradient polyacrylamide slab gels. An aliquot of the total nuclear protein or the SC preparation was dissolved in the sample buffer (containing 62.5 mM Tris-HCl (pH 6.8), 8 M urea, 2% (w/v) SDS and 0.00125% (w/v) bromophenol blue). Samples were loaded onto the gel without heating and subjected to electrophoresis at a constant current of 20 mA for 1.5 h. The gel was stained according to the method described by Wray [23]. Standard proteins used for calibrating M r values were as follows: myosin (200000), /3-galactosidase (116 000), phosphorylase b (97 400), bovine serum albumin (66 200), ovalbumin (42 699), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500) and lysozyme (14 400). For immunoblotting, the total nuclear proteins were separated on a 10% SDS-polyacrylamide slab gel with 4% stacking gel. After electrophoresis, the gel was rinsed in the blotting buffer (containing 0.125 M Tris base, 0.96 M glycine and 20% (v/v) methanol) for 30 min. The proteins in the gel were transferred to a nitrocellulose filter using a semi-

118 dry transblot apparatus at a constant current of 0.8 mA/cm 3 for 2 h. All the incubation steps in immunodetection were performed at room temperature. After blotting, the filter was divided into strips, and the strips were incubated for 2 h in blocking solution containing 1% (w/v) dry milk in TBS-T (containing 50 mM Tris-HC1, pH 7.5, 0.15 M NaC1 and 0.5% Tween-20). The strips were then incubated for 2 h in diluted serum (diluted 1:1000 with blocking solution) with gentle agitation. After washing 3 times in blocking solution for 5 min each, the blots were incubated for 1 h with secondary antibody (goat anti-mouse conjugated with alkaline phosphatase, Kirkegaard & Perry Laboratories Inc., USA, diluted 1:500 with blocking solution). Following several washes in TBS-T, antibodies bound to the filter were detected with 0.33 mg/ml nitro blue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-3-indolylphosphate in alkaline phosphatase buffer (containing 0.1 M Tris-HC1 (pM 9.5), 5 mM MgC12 and 0.1 M NaC1) [24]. Result

Isolation of synaptonemal complexesfrom microsporocytes Lily microsporocytes at pachytene of meiotic prophase, the stage when pairing of the h o m o l -

ogous chromosomes has been completed, were used as a source of the isolation of SCs. The cells were gently homogenized by nitrogen decompression and the nuclei were purified by sedimentation through the series of Percoll density gradients as described in Materials and Methods. Most of the material obtained were the nuclei from meiotic cells, though the nuclei from tapetal cells and fragments of cell wall were found in this fraction as contaminants. The electron micrograph of a thin section of the isolated nucleus showed distinctive SC structures surrounded by masses of condensed chromatin (not shown), indicating that the SCs were preserved with intact morphology even after the several purification steps. The nuclei thus purified were subjected to DNase II treatment for removal of chromatin fibers which protruded from SCs during isolation. Behavior of the nuclei during the course of DNase II digestion was followed under a differentialinterference-contrast microscope (Fig. 1). The nuclei suspended in TED buffer were rapidly swollen and condensed chromosomes were not observed (Figs. la and lb). On incubation with DNase II, thread-like structures became visible (Fig. lc). These structures were released from the nucleus concomitant with degradation of the nuclear structure itself during prolonged DNase II

Fig. 1. Differential-interference-contrastmicrographs of isolated nucleus and DNase-treated nucleus of lily microsporocyte. (a)

Isolated nuclei. (b) A swollen nucleus in TED buffer. (c) A nucleus treated with DNase 11 for 30 min at 20°C. Bar = 10 t~m.

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a

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Fig. 2. Silver and fluorescence staining of isolated nucleus and isolated SCs. A nucleus (a,b~ and isolated SCs (c,d) were stained with silver (a,c) and DAP1 (b,d) and observed under a light microscope as described in Materials and Methods. The arrowhead indicates nucleolus. Bar = 10/~m.

treatment and disappeared from the nucleus at the end of incubation. Figure 2 shows the same process followed by staining the nuclei with silver nitrate and the fluorescent dye, DAPI. Before DNase II digestion, thin threads of SCs, stained with silver, were distributed in every direction in the isolated nucleus (Fig. 2a). As the DNase II treatment proceeded, SCs were broken into fragments with approximate lengths of 10/~m or less (Fig. 2c). The fluorescence of DAPI, on the other hand, gradually reduced in intensity as digestion of DNA proceeded and consequently became hardly visible along the silver-stained SC fragments, which means that most of DNA were removed from the axes of the paired chromosomes (Figs. 2b and 2d). SCs released from the nucleus were separated from

residual nuclear components including degraded chromatin and fragments of the nucleolus by centrifuging through a linear gradient of Nycodenz instead of sucrose employed by Heyting et al. [10] as described in Materials and Methods. To examine the degree of preservation of fine structure, the preparation of SCs were subjected to analysis by means of electron microscopy. While some fragmentation took place, isolated SCs seemed to possess a pair of rod-like structures, a characteristic of lateral elements of SCs in vivo even after purification (Fig. 3b). However, the distance between two lateral elements was wider than that of intact SCs (200 nm vs. 100 nm) and the central element which was supposed to be located in-between the lateral elements became less obvious as chromatin digestion proceeded (Figs.

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. . . . . . . . .

Fig. 3. Electron micrographs of SCs isolated from lily microsporocytes. Isolated nuclei were treated with 200 Kunitz units/ml of DNase II as described in Materials and Methods. (a) A fragment of SC obtained after 1 h incubation with DNase 11. Tufts of remnant chromatin are attached to the lateral elements ofSC. (b) A fragment of SC obtained after 2 h incubation with DNase II. Bar = 1 #m.

b

3a a n d 3b). This might be due to p a r t i a l r e m o v a l o f some m a t e r i a l s between the elements, e.g. D N A strands or c h r o m a t i n , by enzyme treatment.

Antiserum against the isolated SCs The serum was collected from a B A L B / C m o u s e injected with isolated SCs a n d i m m u n o s t a i n i n g o f the meiotic nuclei was carried o u t as described in M a t e r i a l s and M e t h o d s , The a n t i s e r u m raised against the isolated SCs b o u n d to SCs o f p a c h y t e n e nuclei intensively a n d a l m o s t exclusively, while the c o n t r o l serum showed only a faint stain (Figs. 4b a n d 4c). Also, the a n t i s e r u m b o u n d neither to the nuclei before z y g o t e n e not after d i p l o t e n e ( d a t a n o t shown) n o r to the nuclei o f tapetal cells (Figs. 4a a n d 4b, lower right). These results i n d i c a t e d that the SCs isolated in this w o r k m a i n t a i n e d i m m u n o l o g i c a l as well as structural characteristics o f SCs in intact nuclei.

Protein composition of isolated SCs To investigate the p r o t e i n c o m p o s i t i o n o f SCs,

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Fig. 4. Immunoperoxidase staining of pachytene nuclei with anti-SC serum. (a) Fluorescence image of nuclei of microsporocyte and tapetal cells (lower right) stained with DAPI. (b) Immunostaining of (a) with anti-SC serum by light microscopy. Note that the tapetal nuclei were not stained. (c) Immunostaining of pachytene nucleus with control serum. Bar = 10 #m.

S D S - p o l y a c r y l a m i d e gel analysis o f the isolated SCs was carried out. Proteins f r o m two independent p r e p a r a t i o n s o f isolated SCs were resolved on an S D S - p o l y a c r y l a m i d e gel (Fig. 5). Protein c o m -

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1

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4

200_

116_ 97_

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values of 1 4 - 24 kDa, gave bands of high intensity in the SC preparation and were also dominant in the extract of the isolated nuclei (Fig. 5, lane 4). Since all of these proteins co-migrated with purified meiotic histones on an SDS-gel (data not shown), it is most probable that they are histone proteins originating from remnant chromatin fibers on isolated SCs as a result of incomplete DNase II digestion. Proteins with Mr values of 42, 50, 52, 116 kDa and 140 kDa were more highly enriched in the SC fraction than in the extract of the nuclei. These proteins were consistently found in various preparations of SCs and were not major ones in the nucleolar fraction (Fig. 5, lane 3).

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116, 97, 66, ~i~i"~ii!!!!i'!'i~!~i,~ii,~,~i~, ~ -'*-"

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31 Fig. 5. SDS-polyacrylamide gel electrophoresis of proteins in isolated SCs, nucleoli and nuclei fractions. Proteins were extracted from each fraction, resolved on an 8-16% linear gradient gel and stained with silver as described in Materials and Methods. Lanes l and 2, isolated SCs independently prepared; lane 3, nucleolar fraction; lane 4, nuclear fraction. Numbers shown at left are M r values of standard proteins in kDa. Filled circles between lanes 1 and 2 indicate the positions of proteins consistently present in SC preparations.

ponents of the nucleolar fraction obtained from the Nycodenz gradient and of whole extract of the isolated nuclei were also analysed so that possible misinterpretation due to contamination could be avoided. In the SC fractions, several major bands as well as many minor ones were observed (Fig. 5, lanes 1 and 2). Proteins having approximate Mr

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Fig. 6. Immunoblotting analysis of nuclear proteins with antiserum raised against isolated SCs. Total proteins of pachytene nuclei (100 ~g) and proteins of SC fraction (30 p.g) were subjected to SDS-polyacrylamide gel electrophoresis and probetl with normal mouse serum or antiserum, a, Coomassie-bluestained gel; b, immunoblot with normal mouse serum; c, immunoblot with antiserum raised against isolated SCs. Both sera were used at a dilution of 1:1000. In all panels lanes are follows; 1, total proteins of pachytene nuclei; 2, proteins of SC fraction. Numbers shown at left are M r values of standard proteins (kDa). The positions of three antigens with M r values of 52, 50 and 42 kDa are indicated by arrows.

122 Proteins of pachytene nuclei and SC preparation were separated by SDS-PAGE and examined by immunoblotting with antiserum raised against isolated SCs (Fig. 6). The protein profiles of the nuclear extract and isolated SCs stained with Coomassie blue are shown in Fig. 6a. Control serum reacted neither with a blot of nuclear extract nor with an SC blot (Fig. 6b, lanes 1 and 2). When blots were probed with antiserum, three antigens with Mr of 43, 50 and 52 kDa were clearly detected on a nuclear blot (Fig. 6c, lane 1). On the other hand, the antiserum recognized several antigens with Mr of 36, 42, 45, 50 and 52 kDa on the SC blot, even though they exhibited weak signals (Fig. 6c, lane 2). Three of these antigens, M r values of 42, 50 and 52 kDa, seemed to correspond to the proteins enriched in SC fraction (Fig. 5, lanes 1 and 2). The other two antigens with Mr values of 36 and 45 kDa could hardly be detected in the nuclear extract, but were rather enriched in the SC fraction. The antigen Mr of 43 kDa detected on the blot of nuclear extract was not found on the SC blot, however, it might have an antigenic relation to the proteins found in SC fraction. Discussion

In this report, we have described an improved method for isolating SCs from lily microsporocytes. Nuclei were prepared from meiocytes at pachytene, then SCs were released from the nuclei by digestion of chromatin with DNase II and purified by sedimentation through a Nycodenz gradient. Although there was unavoidable contamination from fragments of nucleoli and undigested chromatin fibers, SC preparations thus obtained were pure enough for further biochemical analysis, based on light and electron microscopical studies. Referring to the structure the SC observed in the nucleus, isolated SCs had well preserved lateral elements, while the central element seemed to be lost to a significant extent (Fig. 3b). When DNase II treatment was applied less intensively during isolation, more central element observed clearly in isolated SCs (Fig. 3a). This suggest that DNA is involved in construction of the central element between a pair of lateral elements.

SCs isolated and purified from the nuclei had approximate lengths of 10 ~m or less, which are about one-tenth of the average length of the intact SCs in the lily nucleus (Figs. 2c and 3). By continuous observation of a single nucleus during the DNase II treatment under the light microscope, we found that fragmentation of SCs occurred as digestion progressed, suggesting the presence of preferred sites along SCs for DNase II. It might therefore be plausible that a portion of SCs are formed by tandem connection of shorter segments by double stranded DNA and that DNase II digests such connecting sites which are still accessible to the enzyme and thus causes fragmentation of SCs. Antiserum raised against the isolated SCs clearly and specifically bound to SCs in pachytene nuclei of lily microsporocytes. The antiserum, however, did not react with microsporocytes of rice or oat. Such specificity seems to be surprising since the structure and the dimension of SCs are the same among organisms. One possible explanation is that the universal component(s) of SCs have only low antigenicity and that the antibodies were raised only against minor component(s) which are specific to the organism. Even after an extensive treatment with DNase II during purification of SCs from lily microsporocytes, several distinctive proteins, as well as histones and proteins probably originating from contaminated nucleoli, were present as discrete bands on SDS-gels (Fig. 5). The protein composition of isolated SCs analysed in this study seem to be quite different from that of rat spermatocytes described by Heyting et al. [10]. A series of biochemical and immunochemical studies on SC components of rat spermatocytes [ 11-14,25] have shown that proteins with M r values of 30, 33, 125 and 190 kDa are parts of lateral elements and that a protein with Mr, of 48 kDa is a component of a central element. In yeast, the HOP1 gene product having a molecular weight of 70 kDa has been demonstrated to be a constituent of SCs [26]. In our preparation, however, no obvious band corresponding to either of these proteins was found. Instead, five proteins with Mr values of 42, 50, 52, 116 and 140 kDa were consistently present in various preparations of lily SCs. The antiserum raised against the isolated SCs recognized five

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antigens with Mr values of 36, 42, 45, 50 and 52 kDa present in the SC fraction (Fig. 6). Among these, 42, 50 and 52 kDa antigens correspond to three of the major proteins present in the SC fraction and they therefore seem to be probable candidates for constituents of SCs. The antiserum also reacted with a protein of 43 kDa in the nuclear extract, however, we could not determine its relevance to SCs in this study. Several kinds of monoclonal antibodies against SCs have been isolated using purified lily SCs as antigen and one of them recognizes a nuclear protein with Mr of 36 kDa present exclusively in the nuclei of zygotene and pachytene stage (unpublished results). Intensive studies with these antibodies should lead to identification of the exact constituents of SCs.

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Acknowledgments This work was supported by a grant pioneering research project in biotechnology from Ministry of Agriculture, Forestry and Fisheries of Japan. This work was also supported in part by Grant-in-Aid for Special Research on Priority Areas (Project No. 01660001, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, Japan.

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