FIGURE LEGENDS
Figure 1. Overview of the time series samples of embryo and
endosperms. Dotted line indicates the manual dissection of endosperm,
cutting it into upper and bottom endosperms. DAF, days after
fertilization; E, embryo; En, endosperm; EnB, bottom part of endosperm;
EnU, upper part of endosperm; NB, notched-belly rice mutant; WT, wild
type of Wuyujing-3. Bar = 1 mm
Figure 2. Global transcriptome relationships among tissues and
developmental stages. (a) Venn diagram of the 20820 genes expressed in
the embryo and endosperm. (b) Number of genes detected in each tissue.
(c) Comparison of gene activity between embryo and endosperm. (d) PCA of
the seed tissue mRNA populations. PCA plot shows two distinct groups of
embryo and endosperm mRNA populations: group I for embryo and group II
for endosperm. (e) and (f) Clustered dendrogram showing global
transcriptome relationships of time series samples from the embryo of WT
and NB, respectively. (g) to (h)
Clustered dendrogram showing global transcriptome relationships of the
upper and bottom endosperms of WT, respectively. (i) to (j) Clustered
dendrogram showing global transcriptome relationships of the upper and
bottom endosperms of NB, respectively. The bottom row indicates the
developmental phases according to the cluster dendrogram of the time
series data. Except in (a), data of 5, 10, 15…, and 60 represent
sampling timepoints (days after fertilization).
Figure 3. Expression patterns of genes in different
co-expression modules for embryo and endosperm of WT (a, b) and NB (c,
d). Co-expression modules are ordered according to the sample time
points of their peak expression. For each gene, the FPKM value
normalized by the maximum value of all FPKM values of the gene over all
time points is shown. The numbers of genes and TFs in each module are
shown on the right.
Figure 4. Carbohydrates,
proteins, SnRK1, and hormones, and their regulating genes in the embryo
and endosperm of WTthroughout development. Each value represents the mean ± SE of
three replicates. Black, orange, and blue indicate non-tissue, embryo,
and endosperm specific genes, respectively.
Figure 5. Minerals in the embryo and endosperm of WT and NB
across the developmental stages. Each value represents the mean ± SE of
three replicates.
Figure 6. Diagrammatic representation of the new
comparison method for quantifying the embryo effect on endosperm
development. This method assembles three key components: (a)
Quantification of the position
effect (PE) by comparison between
the upper (WT_EnU) and bottom (WT_EnB) endosperms of WT. This reflects
the difference in position between upper and bottom endosperms within
the WT grain, where nutrients and signals move freely without being
blocked by the notched line as in NB. (b) Quantification of the compound
effect (CE) of position and embryo by comparison between the upper
(NB_EnU) and bottom (NB_EnB) endosperms of NB. Due to the notched
line, movement of nutrients and signals are severely constrained between
the two endosperms. Therefore, the influence of the embryo is trapped
and thus mainly expressed in the bottom endosperm. Note that 2/3
interface between the upper and bottom is cut down by the notched line.
(c) Evaluation of the embryo effect (EE) by secondary comparison of
CE/PE to exclude the position effect. Three metabolites are used to
exemplify the working principle of this method. For example, Asn content
is up-regulated in the bottom endosperm of both WT and NB. By contrast,
comparison of NB and WT reveals that the embryo effect is
down-regulating it in the endosperm. Red and navy blue squares indicate
up and down-regulation in the bottom endosperm, respectively.
Figure 7. Putative role of T6P-SnRK1 signaling in developmental
transition of the endosperm as revealed by the proposed new comparison
method. Effects of position, compound, and embryo are all shown. (a)
Schematic diagram showing the putative T6P-SnRK1 signaling pathway. (b)
Heatmap of differentially expressed genes associated with position,
compound, and embryo effects. ADP-G, ADP-glucose; AGPase, ADP-glucose
pyrophosphorylase; DBE, debranching enzyme; Fru, fructose; G6P/PT,
glucose-6-phosphate/phosphate translocator; G1P, glucose 1-phosphate;
GA, gibberellin; GBSS, granule-bound starch synthase; Glc, glucose; IAA,
indoleacetic acid; INV, invertase; ISA, isoamylase; MST, monosaccharide
transporter; PM, plasm membrane; SBE, starch branching enzyme; SnRK1,
sucrose non-fermenting-1 related protein kinase 1; SSP, seed storage
protein; SSS, soluble starch synthase; SuSy, sucrose synthase; SUT,
sucrose transporter; T6P, trehalose 6-phosphate; TAR2, tryptophan
aminotransferase 2; TPP, trehalose 6-phosphate phosphatase; TPS,
trehalose 6-phosphate synthase; Trp, tryptophan; UDP-G, UDP-glucose;
UGPase, UDP-glucose pyrophosphorylase; α-AMY, α-amylase; β-AMY,
β-Amylase. Red and navy rectangles indicate up- and down-regulation,
respectively.
Figure 8. Holistic and dynamic picture of seed development. (a)
Schematic illustration of the morphological changes in embryo
(longitudinal section) and endosperm (transversal). Varying colors of
the pericarp and testa show the progression of degradation in maternal
tissues, while those of the starch endosperm show the grain-filling
process. (b) Dynamic accumulation of storage materials (sucrose, starch,
FAAs, storage proteins, and minerals) and the increase in grain weight,
length, width, and thickness. (c) Molecular signatures of the embryo and
endosperm at different developmental stages.