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Figure S1. Development of rice syncytial endosperm in WT.
The PI-stained ovary samples at 24 (a and f), 36 (b and g), 48 (c and h), 60 (d and i) and
72 HAP (e and j) were fixed, stained with PI and observed by CLSM. (a–e) and (f–j) are
optical sections showing the median and surface, respectively, of the endosperm. The
arrows in (a) indicate syncytial nuclei around the central vacuole. The inset (i’) shows
an abnormally enlarged nucleus, which was occasionally observed in the WT syncytium
(see text). Scale bars: 100 µm. cv, central vacuole; and em, embryo.
Figure S2. CLSM images showing a mitotic nucleus with an excessive number of
chromosomes in the enl1 endosperm at 24 HAP.
(a) Z-stack image of the entire nucleus. (b) A single optical section that constitutes the
Z-stack image in (a). Scale bars: 20 µm.
Figure S3. CLSM optical sections of PI-stained shoot apices.
(a) WT. (b, c) enl1. The nuclear morphologies in the enl1 shoot apices including shoot
apical meristems (arrows), leaf primordia and developing leaves were usually
indistinguishable from those of WT (a, b). However, extended nuclear aggregates were
found in a single observation (right square brackets in c). Scale bars: 20 µm.
Figure S4. Molecular identification and characterization of ENL1.
(a) Schematic illustration for the mapping of the enl1 locus to the chromosome 4
long-arm terminal region. The initial mapping revealed a linkage between the marker
HKM1 and the endospermless phenotype among the segregating F2 seeds (top). Fine
mapping using the F2 and F3 populations localized enl1 to the telomeric side of the
SNP24 marker (middle). The numbers of recombinations are indicated between the
marker positions. The dashed line in the middle row map indicates the unsequenced
genomic region between the BAC clones AL606656 and AP009057 in the RAP-DB
build 5. The AP009057 sequence containing the telomere was highly rich in repeat
sequences. Recombinants with breakpoints beyond the SNP6 marker toward the
telomere were not identified. Thus, all of the exons and exon-intron boundaries of the
genes, which were annotated in the region between marker SNP24 and the end of
AP009057 and were potentially related to the phenotype, were sequenced using the enl1
plant genomic DNA. A mutation was found only in LOC_Os04g59624 as indicated
(bottom schema in (a), depicted as a blue arrow). The arrows represent annotated ORFs
in RAP-DB. The gray arrow represents LOC_Os04g59620, which is a paralog of
LOC_Os04g59624. (b) Comparison of the nucleotide sequences of the third exon of
LOC_Os04g59624 between WT (upper letters) and enl1 (lower letters). The alignment
revealed the presence of a T->A substitution and a nearby small rearrangement that
resulted in the loss of 11 bases and several base changes in enl1. These sequence
changes created a premature stop codon in this exon of enl1. (c) Schematic
representation of the gene structures of LOC_Os04g59624 and LOC_Os04g59620. The
twenty exons of the former and the latter are represented by blue and gray boxes,
respectively. The 5’- and 3’-UTRs are depicted with open boxes. (d) Gene constructs
that were used to produce transgenic plants. tgENL1 comprises the native promoter
(black bold line) and gene body of LOC_Os04g59624 (thin line with blue and white
boxes representing exons). The XbaI site that was artificially created in the 7th intron
for genotyping is shown by an asterisk. tgENL1-Venus has a Venus-coding sequence
(green box) that is fused to the coding sequence of the last exon of tgENL1.
pUbi::tcENL1-Venus comprises the maize ubiquitin promoter (yellow bold line) that is
fused to the LOC_Os04g59624 cDNA (coding sequence of AK242828, blue box)
followed by the Venus-coding sequence. pENL1::tcENL1-Venus has the native promoter
of LOC_Os04g59624 in place of the maize ubiquitin promoter in pUbi::tcENL1-Venus.
pENL1::tcENL1L-Venus comprises the native promoter of LOC_Os04g59624 that is
fused to the cDNA from LOC_Os04g59620 in a flipped orientation (coding sequence of
AK318609, gray box; note that the annotation of this cDNA clone in the databases is
made for the reverse orientation) followed by the Venus-coding sequence.
pENL1L::tcENL1L-Venus has the intergenic region between LOC_Os04g59624 and
LOC_Os04g59620 (gray bold line) as the promoter in place of the LOC_Os04g59624
promoter in pENL1::tcENL1L-Venus.
Figure S5. ENL1 protein levels fluctuate with cell cycle progression.
(a) Total proteins were extracted from the WT and enl1 seedlings and analyzed by
western blotting using the anti-ENL1 antibody. The proteins were blotted onto a
nitrocellulose membrane and detected by pre-staining with the MemCode staining
solution (left) or immunologically (right). The ENL1 protein (116 kD) and its putative
degradates were detected in the WT protein extract using the anti-ENL1 antibody. The
absence of cross-reacting bands in the enl1 extract verified the specificity of the
antibody. (b) Changes in the proportions of the 2C (blue line) and 4C cells (pink line) in
the synchronized rice cultured cells (line Oc) after release from G1 arrest. (c) Changes
in the ENL1 protein levels in the synchronized cells that were examined in (b) were
analyzed by western blotting with a loading control by pre-staining the blotted
membrane with MemCode. The ENL1 protein levels appeared to increase at S phase
toward M phase.
Figure S6. Disruption of Arabidopsis CHR24 has marginal and no apparent effects on
endosperm development and seedling growth, respectively.
(a) T-DNA insertions in the Arabidopsis ENL1 ortholog CHR24. (b) CLSM image of the
PI-stained endosperm in the WT (Col-0, top) and SALK_152488 line (bottom);
although infrequently, nuclei carrying multiple nucleoli could be observed in
SALK_152488 endosperms (arrows in right bottom), but their development appeared
normal. (c) Seedlings of WT, SALK_152488 (left) and SALK_050793 (right) ten days
after germination. Scale bars: 30 µm in (b, lefts); 5 µm in (b, rights); and 2 cm in (c).
Table S1. Complementation of enl1/+ in T0 plants.
Table S2. Complementation of enl1 in T1 plants.
Table S3. Primers that were used for mapping.
Table S4. Other primers that were used in this study.
Methods S1. Supporting experimental procedures.