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\begin{document}

\title{Integration of embryo-endosperm interaction into a holistic and dynamic
picture of seed development using a rice mutant with notched-belly
grains}



\author[1]{Yang Tao}%
\author[1]{Lu An}%
\author[1]{Feng Xiao}%
\author[1]{Ganghua Li}%
\author[1]{Yanfeng Ding}%
\author[2]{Matthew J. Paul}%
\author[1]{Zhenghui Liu}%
\affil[1]{Nanjing Agricultural University}%
\affil[2]{Rothamsted Research}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Embryo-endosperm interaction strongly affects the developmental process
of seeds and hence the formation of crop yield and quality. However,
signals that orchestrate developmental processes of the two compartments
are largely unknown. This study aims to provide an integrative landscape
of rice seed development by organizing the information about
embryo-endosperm interaction. Using a mutant with notched-belly grains,
we devised a novel comparison method that can quantitatively evaluate
the effect of embryo on the developmental process of endosperm, by
comparing the upper and bottom endosperms of the mutant. Global
comparison of mRNAs datasets in embryo and endosperm samples collected
from 5 DAF (day after fertilization) through maturity uncovered a
dragging effect of embryo on the developmental transition of the
endosperm, extending the middle stage and delaying the late stage.
Biochemical analysis revealed that signaling pathways of T6P-SnRK1, and
GA or IAA may be involved in mediating this developmental transition.
Integrating the information about embryo-endosperm bidirectional
dialogue enables a new rice seed developmental staging system to be
proposed. The results obtained should be valuable for elucidating the
biological processes and signatures of grain formation, hence providing
a valuable resource for modifying the molecular and physiological
processes responsible for cereal grain yield and quality.%
\end{abstract}%



\sloppy


\textbf{Integration of embryo-endosperm interaction into a holistic and
dynamic picture of seed development using a rice mutant with
notched-belly grains}

Yang Tao\textsuperscript{1}, Lu An\textsuperscript{1}, Feng
Xiao\textsuperscript{1}, Ganghua Li\textsuperscript{1}, Yanfeng
Ding\textsuperscript{1}, Matthew J. Paul\textsuperscript{2}, Zhenghui
Liu\textsuperscript{1, 3, *}

\textsuperscript{1} College of Agriculture, Nanjing Agricultural
University, Nanjing 210095, China

\textsuperscript{2} Plant Science, Rothamsted Research, Harpenden,
Hertfordshire AL5 2JQ, UK

\textsuperscript{3} Collaborative Innovation Center for Modern Crop
Production, Nanjing Agricultural University, Nanjing 210095, China

*To whom correspondence may be addressed: Zhenghui Liu, Email:
liuzh@njau.edu.cn



\textbf{Abstract}

Embryo-endosperm interaction strongly affects the developmental process
of seeds and hence the formation of crop yield and quality. However,
signals that orchestrate developmental processes of the two compartments
are largely unknown. This study aims to provide an integrative landscape
of rice seed development by organizing the information about
embryo-endosperm interaction. Using a mutant with notched-belly grains,
we devised a novel comparison method that can quantitatively evaluate
the effect of embryo on the developmental process of endosperm, by
comparing the upper and bottom endosperms of the mutant. Global
comparison of mRNAs datasets in embryo and endosperm samples collected
from 5 DAF (day after fertilization) through maturity uncovered a
dragging effect of embryo on the developmental transition of the
endosperm, extending the middle stage and delaying the late stage.
Biochemical analysis revealed that signaling pathways of T6P-SnRK1, and
GA or IAA may be involved in mediating this developmental transition.
Integrating the information about embryo-endosperm bidirectional
dialogue enables a new rice seed developmental staging system to be
proposed. The results obtained should be valuable for elucidating the
biological processes and signatures of grain formation, hence providing
a valuable resource for modifying the molecular and physiological
processes responsible for cereal grain yield and quality.

\textbf{Keywords} : Seed development; embryo-endosperm interaction;
transcriptome; sugar signaling; rice physiology



\textbf{INTRODUCTION}

The seed (grain) of rice (\emph{Oryza sativa} ) is a complex but
delicate biological system, containing three genetically distinct
tissues, the diploid embryo, the triploid endosperm, and the diploid
maternal tissues (An et al., 2020). Rice is a main contributor of
dietary calories and nutrients for nearly half of the global population
(Wu, M\selectlanguage{ngerman}üller, Gruissem, \& Bhullar, 2020). Rice grain yield has to be
sustainably improved both in quantity and quality, to solve formidable
challenges including the ever-increasing population, climate change, and
the quest for high-quality rice that has accompanied the rise in living
standards (Sreenivasulu et al., 2015). A detailed understanding of the
molecular and physiological mechanisms underpinning seed development may
enable the design of effective strategies to boost rice quality and
yield.

Milled rice is the main form of rice for consumption produced by milling
during which the outer maternal layers, the embryo, the aleurone, and
part of the starchy endosperm are removed. End use quality of rice seed
is dominated by the physico-chemical properties of endosperm that are
predominately packed with starch granules and protein bodies (Xi et al.,
2014). Grain chalkiness affects grain quality due to perturbation of
carbohydrate metabolism (Nakata et al., 2017) or protein synthesis
(Ishimaru et al. 2019; Wada et al., 2019). Up to now, the majority of
research on rice quality has been centered on the starchy endosperm,
revealing that it is a rather sophisticated trait controlled by multiple
genes and by environmental conditions (Fitzgerald, McCouch, \& Hall,
2009; Ishimaru et al., 2019; Lin et al., 2017).

The composition, structure, and size of endosperm are affected by its
interactions with other tissues, in particular the embryo, during the
whole process of seed development. Endosperm provides nutrients to drive
embryo growth, and physically restrains its size and development. The
embryo is the germline component of the seed, and affects the allocation
of sugars and other nutrients like amino acids and minerals to the
endosperm. Concomitant development of embryo and endosperm under the
constraint of maternal tissues (seed coat or pericarp) requires
coordination of the two compartments (Doll et al., 2020; Lafon-Placette
\& Köhler, 2014). Growing evidence in rice as well as Arabidopsis
(\emph{Arabidopsis thaliana} ) and maize (\emph{Zea mays} ) supports
bidirectional communication between embryo and endosperm, as reviewed by
Lafon-Placette and Köhler (2014); An et al. (2020) and Ingram (2020).
Therefore, both embryo and endosperm and their interaction affect grain
filling and quality.

Transcriptome analyses of seed development have already identified most
genes expressed in the different compartments (Olsen, 2020). These
comprehensive studies provided insights into the molecular networks and
pathway interactions that function during the development of individual
seed compartments for Arabidopsis (Belmonte et al., 2013), maize (Chen
et al., 2014; Yi et al., 2019), wheat (\emph{Triticum aestivum} ; Xiang
et al., 2019), and barley (\emph{Hordeum vulgare} ; Bian et al., 2019).
In rice, Ishimaru et al. (2019) isolated the aleurone, dorsal, central
and lateral tissues of developing endosperm by laser-capture
microdissection (LCM), and profiled the gene expression by a 44 K
microarray, revealing that high molecular weight heat shock proteins
have a role in mediating redox, nitrogen and amino acid metabolism in
the chalky tissue under heat stress. Wu et al. (2020) profiled gene
expression activity in the nucellar epidermis, ovular vascular trace,
endosperm and the aleurone at three distinct grain development stages,
offering insights into the molecular aspects of grain development. Ram
et al. (2020) employed an rapid LCM approach to individually collect
pericarp, aleurone, embryo and endosperm at 10 DAF (days after
anthesis). Subsequent RNA-Seq analysis identified 7760 differentially
expressed genes, by which the authors built some key tissue-specific
pathways responsible for nutrient partitioning.

However, these studies do not enable an integrated understanding of the
cellular processes governing the formation of rice grain, because of the
scarcity of information on embryo-endosperm communication and its
influence on rice yield and quality, which is partly attributed to the
complexity of seed structures (Doll et al., 2020).

Notched-belly grain is a misshapen type of rice grain, having a
notched-line on its ventral side and thus being inferior in appearance
and milling quality (Tong et al., 2018). Previously, we identified a
notched-belly mutant (NB) of a japonica rice Wuyujing 3 using a chemical
mutagen (Lin et al., 2014). It has high occurrence of white-belly
grains, and the notched line is visible at 5 DAF, which separates the
endosperm into two sub-compartments, the translucent upper part and the
chalky bottom. Using this mutant, we developed a novel comparison system
that can evaluate the embryo effect on endosperm composition, with a
conclusion that embryo demonstrated a negative effect on the contents of
proteins, amino acids, and minerals in the chalky endosperm (Lin et al.,
2016). Complementary proteome and transcriptome profiling of the NB
mutant suggested the transporters for metabolites and the signal
messengers of hormones as potential signals for embryo-endosperm
communications, confirming the value of the NB mutant for examining the
role of embryo in grain filling and quality formation (Lin et al.,
2017).

In this study, we used the NB mutant to characterize the genome-wide
gene expression profile of embryo and endosperm samples between 0-60
DAF. In combination with physiological and histological investigations,
we aimed to: (i) unravel the commonalities and specificities between
embryo and endosperm in developmental processes as well as molecular and
metabolic signatures; (ii) manifest the effect of embryo on endosperm
development; and (iii) frame a holistic and dynamic picture of seed
development by integrating the information of embryo-endosperm
crosstalk. Comprehensive comparison of mRNAs datasets revealed a
dragging effect of embryo on the developmental transition of the
endosperm, which might be mediated by hormonal (GA and IAA) and
T6P-SnRK1 signaling pathways. A new staging system is subsequently
proposed by integrating the information about embryo-endosperm
bidirectional communications. The findings obtained should offer
insights into the biological processes and signatures of grain
formation, hence providing a valuable resource for the manipulation of
molecular and physiological processes responsible for cereal grain yield
and quality.

\par\null

\textbf{MATERIALS AND METHODS}

\textbf{Plant materials and sampling}

The notched-belly mutant (NB) was obtained by EMS treatment of a
japonica rice cultivar Wuyujing3 (WT). It possesses a high ratio of
notched-belly grains with a white-belly mainly on the bottom endosperm
(Lin et al., 2014). In 2018, six seedlings of WT and NB were
transplanted into a plastic pot filled with 10 kg paddy soil. The plants
were grown under natural conditions, and at two days before flowering
were transferred to a 31\selectlanguage{english}/24\selectlanguage{english} growth chamber (12h/12h day and night
cycle), with light intensity of 600 \selectlanguage{greek}µ\selectlanguage{english}mol photons
m\textsuperscript{-2}s\textsuperscript{-1} and relative humidity 70 \selectlanguage{ngerman}±
5\%. Flowering dates were carefully marked for the sampled caryopsis on
the middle primary rachis. Grains were sampled at eight times, i.e. 5,
10, 15, 20, 25, 30, 45 and 60 DAF, with three biological replicates.
Samples were quickly frozen in liquid nitrogen and then stored at -80 \selectlanguage{english}
until analysis. The developing grains were manually dissected into three
subsamples, the embryo, and the upper and bottom part of the endosperm
(Figure 1). It is noteworthy that the endosperm samples contain the
maternal tissues such as pericarp and seed coat.

\textbf{Gene expression profiling using RNA-Seq}

About 0.1 g sample was used for total RNA extraction by TRIzol reagent
(Invitrogen, USA). The extracted RNA was dissolved in 100 \selectlanguage{greek}μ\selectlanguage{english}l of
RNase-free water, and then quantified using a NanoDrop spectrophotometer
(Thermo Scientific, USA). RNA quality was evaluated using the 6000 Pico
LabChip of the Agilent 2100 Bioanalyzer (Agilent, USA). The
qualification and quantification of the sample library were performed
using an Agilent 2100 Bioanaylzer and StepOnePlus Real-Time PCR System
(Applied Biosystem, USA). The library products were sequenced with
BGISEQ-500 (BGI, China). Nipponbare reference genome (IRGSP-1.0;
https://www.ncbi.nlm.nih.gov) was used to process the RNA-Seq libraries
into read mapping and analysis. To ensure good quality and effective
mapping, the low-quality reads were removed using the SOAPnuke (V1.4.0)
and trimmomatic (V0.36) software (Chen et al., 2018b), and only clean
reads were processed for mapping using HISAT2 (V2.1.0) (Kim, Langmead,
\& Salzberg, 2015). The gene expression level was measured by RSEM
(V1.2.8) software (Li \& Dewey, 2011), and fragments per kilobase of
transcript per million mapped reads (FPKM) values were used for
quantifying gene activity. We only considered a gene as expressed if its
FKPM value [?] 1 to inhibit the influence of transcriptomic noise. To
confirm the accuracy and authenticity of the three biological repeats,
we calculated the Pearson correlation coefficient among them with the
normalized expression level of log\textsubscript{2} (FPKM value +1)
(Figure S1). To identify differentially expressed genes (DEGs), we
considered a DEG when it exhibits an absolute value of
log\textsubscript{2} ratio [?]1 compared with an FDR corrected P-value of
[?]0.001 (Wang, Feng, Wang, Wang, \& Zhang, 2010).

\textbf{Validation of transcriptome data using qRT-PCR}

RNA-Seq data quality was validated by measuring the relative expression
pattern of 12 genes functioning in C and N metabolism using qRT-PCR
(Figure S2). Briefly, target-specific qRT-PCR primers were designed
using the Primer5.0 software and synthesized, as presented in Table S1.
First-strand cDNA was synthesized with purified total RNA using the
PrimeScript\textsuperscript{TM} RT reagent Kit (Takara, Dalian, China).
qRT-PCR was carried out in Lightcycler 480II (Roche, Rotkreuz,
Switzerland) using 2X SG Fast qPCR Master Mix (B639271, BBI, Roche).
Each sample was represented by three biological and three technical
replicates. The comparison of qRT-PCR results with RNA-Seq revealed the
similar expression pattern between two datasets, thus validating the
transcriptomic data (Figure S2). In addition, transcript abundance
pattern of embryo (\emph{OSH1} and \emph{OsLEC1} ) and
endosperm\emph{(GluD-1} and \emph{RPBF)} specific genes also confirmed
the credibility of the gene expression data (Figure S3), indicating that
the embryo and endosperm samples were processed well.

\textbf{PCA and hierarchical clustering}

To facilitate graphical interpretation of relatedness among the 48
tissue samples, we reduced the dimensional expression data to two
dimensions by PCA through Omicshare
tools\emph{(https://www.omicshare.com/tools/Home/Soft/pca)} .
Hierarchical clustering (HCL) was carried out through Omicshare tools
(\emph{https://www.omicshare.com/tools/Home/Soft/hca} ).

\textbf{Gene coexpression and functional enrichment analysis}

The co-expression analysis for genes in embryo and endosperm was
conducted using MeV (Howe et al., 2014). Notably, we used the Z-score
value to calculate the relative expression levels before running the MeV
for a tissue. These Z-score values were used as input for MeV and each
individual dataset was clustered using the k-means method and Pearson
correlation coefficients among the genes. Eventually, through Gene
Ontology (GO) annotation (Ashburner et al., 2000), genes were annotated
into the functional categories, and functional enrichment analysis was
performed using the phyper function in R software with default setting
(R Core Team, 2013). P-value was used as a filter mode, and significant
GO categories (P-value \textless{}0.01) were identified. In addition,
embryo or endosperm specific genes were identified by Z-score value. A
gene was declared as embryo or endosperm specific if it had a Z-score
above 2 in at least one of the embryo or endosperm samples as compared
to all the same kind samples.

\textbf{Light microscopy (LM) observation of grain structure}

Grains samples were fixed immediately in 0.1 M phosphate buffer solution
(pH 7.2), containing 2.5\% glutaraldehyde (v/v) and 2\% paraformaldehyde
(w/v). Afterwards, they were dehydrated in a graded ethanol series,
transferred to acetone, and embedded in low-glutinosity Spurr's resin
(Spurr Low-Viscosity Embedding Kit, Sigma-Aldrich, St. Louis, USA). The
sample blocks were polymerized at 70\selectlanguage{ngerman}°C for 8 h. Semi-thin sections (2
\selectlanguage{greek}μ\selectlanguage{english}m) were cut at the longitudinal plane (embryo) and the transversal
plane (endosperm) of the grains, using a Leica Ultrathin Microtome (EM
UC7, Germany). Samples were stained with 1 \% methyl violet, observed
and photographed under a BX53 light microscope (Olympus, Tokyo, Japan).

\textbf{Chemical analysis for metabolites}

Sucrose, fructose, glucose, and starch : Sugar contents were measured by
ultra-high performance liquid chromatography system (UHPLC) as described
by Chen et al. (2018a). Sucrose and hexoses were extracted with 80\%
(v/v) ethanol for 30 min at 80 \selectlanguage{ngerman}°C, followed by centrifugation at 5000g
for 15 min. The supernatant was pooled and lyophilized using a Maxi-Dry
Lyo instrument (Heto-Holten, France), and resuspended in ultrapure water
and filtered through a 0.45 \selectlanguage{greek}μ\selectlanguage{english}m pore size filter. The resulting filtrate
was loaded into a Dionex Ultimate 3000 chromatography system. Starch
content was measured using anthrone reagent at wavelength of 620 nm,
after extraction by ethanol and digestion by perchloric acid.

T6P and SnRK1 activity : T6P was extracted and purified according to the
method of Delatte et al. (2009), and quantified by ultra-performance
liquid chromatography (ACQUITY UPLC I-Class PLUS, Waters, USA) coupled
with triple quadruple mass spectrometry system (Xevo TQ-S micro, Waters,
USA), following the method of Sastre Tora\selectlanguage{ngerman}ño et al. (2012). SnRK1 was
extracted and its activity was assayed by the method of Zhang et al.
(2009), using AMARA peptide as substrates.

Amino acids and proteins : Samples were boiled with 80\% ethanol until
totally bleached. Then, the total volume was kept constant with
distilled water and was centrifuged for 15 min. The supernatant was
pooled and filtered through a 0.45 \selectlanguage{greek}μ\selectlanguage{english}m pore size filter, and then used
for amino acid determination by amino acid analyzer (L-8900, Hitachi,
Japan). Protein fractions including albumin, globulin, prolamin and
glutelin were extracted and measured by the methods of Ning et al.
(2010).

Phytohormones and minerals : IAA, ABA, CKs (tZ and tZR) and GAs
(GA\textsubscript{1}, GA\textsubscript{3}, GA\textsubscript{4}, and
GA\textsubscript{7}) were extracted and purified according to the method
of Wu et al. (2016). IAA, ABA and CKs were quantified by
ultra-performance liquid chromatography (ACQUITY UPLC H-CLASS, Waters,
USA) according to Chou, Chen, Huang, Yu, and Liao (2000). GAs were
quantified by ultra-high performance liquid chromatography (AGLIENT1290,
Agilent Technologies, USA) coupled with mass spectrometry system
(SCIEX-6500Qtrap, AB SCIEX, USA). Minerals, including P, K, Ca, Na, Mg,
Fe, Zn, Mn, and Cu, were quantified by Inductively Coupled Plasma
Optical Emission Spectrometry (ICP-OES 710, Agilent Technologies, USA),
according to the method of Wang et al. (2018).

\textbf{Statistical analysis}

The data of metabolites presented in the study are averages of
triplicate observations. Statistical analysis was performed with SPSS
(V19.0, IBM) statistical software. Multiple comparisons were operated by
Duncan's multiple range test (P \textless{} 0.05). Origin 9.0 was used
for plotting.

\textbf{RESULTS}

\textbf{The generation and analysis of transcriptome data of developing
embryo and endosperm}

To systematically investigate the dynamics of the rice seed
transcriptome over development, we generated RNA-Seq libraries of three
different seed tissues of the wild type WYJ3 (WT) and its notched-belly
mutant (NB) from 8 different time points, including 16 embryos, 16 upper
endosperms and 16 bottom endosperms (Figure 1). In total, we detected
20820 genes expressed in at least one of the 48 samples. Among them,
18171 genes were expressed in both embryo and endosperm, while only 1969
and 680 genes were specifically expressed in embryo and endosperm,
respectively (Figure 2a). Subsequently, the mRNA datasets from different
tissues were compared to get a more specific look into the individual
characteristics of the embryo and endosperm during seed development.
Results showed that most of genes are expressed in seed tissues during
the early phase of seed development (Figure 2b). Within endosperm, both
genotypes showed higher gene activity in upper part of endosperm (EnU)
than the bottom part (EnB). In addition, embryo samples had higher
number of expressed genes as compared to those of the endosperm
throughout seed development (Figure 2c).

\textbf{Categorizing developmental processes of embryo and endosperm
based on gene expression pattern}

To further investigate the relationship between developmental stages and
gene expression pattern, we performed PCA on the complete dataset of
embryo and endosperm (Figure 2d). PCA distinguished the samples into two
distinct groups based on tissue identity, validating the purity of our
tissue samples. Particularly, the first component (PC1; 77.1\% variance
explained) clearly separated the embryo from endosperm samples, while
the second component (PC2; 12.6\% variance explained) discriminated the
stages of development for all of the tissues.

As the PCA proposed the specificity of gene expression across the
tissues and developmental stages, we further classified the
transcriptomic data into a dendrogram by hierarchical clustering (HCL)
analysis, and uncovered four distinguishing groups within embryo and
endosperm, with each group corresponding to a specific developmental
stage (Figure 2e-h).

For embryo samples, both genotypes showed an almost identical dendrogram
(Figure 2e, f). The first cluster is formed at 5 DAF, representing the
stage around differentiation (Em-S1). During this stage, coleoptile and
SAM emerge and first leaf primordium becomes visible on the opposite
side of the coleoptiles, as visualized by electron microscopy (Figure
S4). Similarly, another adjacent cluster from 10-15 DAF represents the
embryo enlargement stage (Em-S2), as characterized by the formation of
second and third leaf primordia, and organ enlargement (particularly the
scutellum). A third cluster at 20DAF corresponds to the maturation phase
(Em-S3) that shows no obviously morphological change. The fourth cluster
from 25 DAF to 60 DAF represents the period of seed dormancy (Em-S4).

By contrast, the duration of developmental stage in endosperm varied
with genotypes. Firstly, endosperms from WT showed four primary clusters
(Figure 2g, h), but those from NB showed only three clusters, suggesting
abnormal development during middle and later stages (Figure 2i, j).
Secondly, within the endosperm, the upper and bottom parts of WT showed
identical duration. By contrast, for NB, the time span of endosperm
filling in the bottom endosperm (NB\_EnB) was prolonged (15-25 DAF), as
compared to the upper endosperm (NB-EnU) that had the identical duration
of second cluster (15-20 DAF) to the WT. Consequently, the maturation
and dormancy stage were delayed in the bottom endosperm and displayed as
a merged cluster (30-60 DAF) of stages 3 and 4, as compared to the upper
part, where stages 3 and 4 cluster had the same duration (25-60 DAF) as
WT. Collectively, these findings indicate a disturbance in the
developmental process of the bottom endosperm of NB by its proximity to
the embryo.

Therefore, normal type of WT instead of the disturbed NB is used for a
general description of the developmental signatures of rice grain. The
four clearly defined clusters in WT endosperm showed that the
differentiation (En-S1) stage last from 5-10 DAF in which it completes
the cellularization, with the aleurone cells starting to become
morphologically distinct from the starchy endosperm (Figure S4). Then
storage accumulation stage (En-S2) occurs at 15-20 DAF, followed by the
maturation (En-S3) phase of the endosperm at 25-30 DAF. The biological
processes involved in dormancy (En-S4) are induced from 45-60 DAF.
Altogether, these results suggested a divergent staging mechanism of
developmental characteristics between embryo and endosperm based on
transcriptomic data.

\textbf{Coexpressed gene sets of embryo and endosperm development}

Using transcript datasets of WT, tissue-specific genes were clustered
into co-expression modules by the k-means clustering algorithm (Howe et
al., 2014). Gene ontology (GO) annotation was performed to assign genes
to functional categories for each module (Ashburner et al., 2000).
Twenty-two coexpression modules were generated for both embryo and
endosperm (Figure 3a, b). Among these modules, 11 and 9 modules were
expressed broadly at more than one stage in embryo and endosperm,
respectively, indicating some common cellular processes across several
stages. Overall, genes from the 11 modules of embryo and the 13 modules
of endosperm were more prevalent at one out of the four developmental
stages, indicating specific function of these modules at the
corresponding stage.

\emph{Cellular processes in the developing embryo}

Cellular processes characterizing each developmental stage were
identified by GO terms that were overrepresented at particular
coexpression modules (Figures 3a, S5a). The early stage of Em-S1, best
represented by modules DP1-DP5, is typified by the overrepresentation
for GO term Ran GTPase binding, mitotic cell cycle, nuclear division,
nuclear DNA replication, cell wall organization or biogenesis, cell
differentiation, mitochondrial protein complex and tricarboxylic acid
cycle. Em-S2 featured modules DP6 and DP7 that were enriched in GO term
characteristic of embryo enlargement, including the cell division, cell
wall organization or biogenesis and lipid droplet. The module DP8
presented an up-regulation of genes involved in nutrient reservoir
activity and response to external biotic stimulus, suggesting the
maturation of embryo characterized by Em-S3. Stage Em-S4, represented by
modules DP9-DP11, was characterized by the overrepresentation of genes
involved in response to biotic stimulus (heat and light), structural
constituent of ribosome, mitochondrial matrix, vacuole organization and
photomorphogenesis. In addition, some genes expressed broadly in embryo
across the sampled time points. All such genes were included in modules
DP12-DP22, and were mostly related to protein complex, Golgi apparatus,
protein kinase activity, cellular response to stimulus, RNA
modification, ribosome biogenesis and endonuclease activity.

\emph{Cellular processes in the developing endosperm}

The functional characterization of endosperm was found different from
that of embryo as different number of modules presented a particular
growth stage (Figures 3b, S5b). Modules DP1-DP8 were characterized as
the stage of En-S1, including genes related to mitotic cell cycle, DNA
replication, cell wall organization, mitochondrion, tricarboxylic acid
cycle and starch biosynthetic process. En-S2 featured the
overrepresentation of GO term nutrient reservoir activity and
transporter activity in D9. Modules DP10-DP12 presented high expression
of genes responsible for response to biotic stimulus (heat and water
deprivation) and lipid storage, representing endosperm maturation at
stage En-S3. Finally, the module DP13 presented an up-regulation of the
genes involved in positive regulation of autophagy, organelle
disassembly, metal ion transport, and cellular catabolic process,
characteristic of the desiccation and dormancy stage of endosperm
(En-S4). Notably, genes responsible for metal cluster binding,
macromolecular complex, RNA processing and signal transducer activity
were overrepresented in DP14-DP22 and were broadly expressed throughout
development.

\textbf{Metabolic dynamics of embryo and endosperm development}

\emph{Sugars and starch}

During embryo and endosperm development, sucrose content gradually
increased, peaking at 15 DAF and then decreased thereafter (Figures 4a,
S6a). Glucose and fructose levels decreased during seed development
(Figures S7a, S7b, S8a, S8b). The ratio of glucose to sucrose mediates
endosperm differentiation (Olsen, 2020). It was higher in the embryo and
endosperm at 5-10DAF, and then decreased gradually (Figures S7c, S8c),
corresponding to the transition from differentiation to storage
accumulation. These sugars were unevenly distributed in rice seed, with
the embryo generally having higher contents (Figures 4a, S6a, S7a, S7b,
S8a, S8b). Genes participating in sugar metabolism were differentially
expressed between embryo and endosperm. For the five genes encoding
sucrose synthase, two of them (\emph{OsSUS1} and \emph{OsSUS6} ) were
mainly expressed in embryo, while another three (\emph{OsSUS2}
,\emph{OsSUS3} , \emph{OsSUS4} ) in endosperm (Figures 4a, S6a).

During grain filling, starch content increased gradually, peaking at 20
DAF and 30 DAF in the embryo and endosperm, respectively (Figures 4b,
S6b). Compared with embryo, endosperm contained higher starch content,
which is consistent with previous report (Juliano \& Bechtel, 1985).
Starch synthesis-related genes were coordinately expressed in the embryo
and endosperm, similar to that in wheat (Xiang et al., 2019). Moreover,
these genes were primarily expressed in endosperm at 5-20 DAF. By
contrast, they displayed a gradual increase in embryo at 5-20 DAF.

\emph{T6P and its target protein} SnRK1

Trehalose-6-phospate (T6P) signals the availability of sucrose in plant
cells through the feast-famine kinase, SnRK1, which targets the genes
responsible for metabolism, growth, and development (Dingkuhn et al.,
2020; O'Hara, Paul, \& Wingler, 2013; Paul, Oszvald, Jesus, Rajulu, \&
Griffiths, 2017). To better understand the role of this signaling
pathway in rice seed, we analyzed T6P content and SnRK1 activities
(Figures 4c, 4d, S6c, S6d). T6P level in embryo and endosperm showed a
gradual decrease in the development course. Compared with endosperm,
embryo has higher content at early stage (5-10DAF) (Figures 4c, S6c).
For example, at 5 DAF, T6P was 3.98 and 6.61 nmol
g\textsuperscript{-1}DW in endosperm and embryo, respectively. In
addition, \emph{TPS} genes were expressed in both embryo and endosperm,
being predominantly expressed at 5-20 DAF.

The SnRK1 activity in endosperm maintained a low level at 5-20 DAF, and
then increased rapidly at later stages (25-60 DAF). Different from the
endosperm, SnRK1 in the embryo was at a lower level, being higher only
at 5 DAF (Figures 4d, S6d). \emph{SnRK1A} was specifically expressed in
the embryo, while two \emph{SnRK1B} genes (\emph{SnRK1B} and\emph{OSK35}
) were mainly expressed in endosperm. In addition,\emph{SnRK1A} was
broadly expressed across the time points in embryo. By contrast,
\emph{SnRK1B} and \emph{OSK35} were more prevalent in endosperm at 5-20
DAF. Interestingly, in contrast to the strong correlations between the
actual level of T6P and the activity of its regulating genes, those for
the SnRK1 activity showed no significant correlation, indicating a
strong post-translational regulation.

\emph{Amino acids and proteins}

During embryo development, the level of total free amino acids (FAAs)
gradually decreased at 5-25 DAF and increased thereafter (Figures S7d,
S8d). In contrast, FAAs in endosperm decreased as grain filling
progressed. In addition, FAAs was higher in embryo than endosperm.
Storage proteins were higher in embryo than endosperm (Figures 4e, S6e).
However, gene expression pattern of storage protein synthesis showed an
opposite correlation with storage proteins content, suggesting that
their encoding genes are specifically expressed in the endosperm, as in
wheat (Xiang et al., 2019).

\emph{Phytohormones}

GAs showed a gradual decrease over seed developmental course. Compared
with endosperm, embryo had higher content at 5 DAF, while exhibited an
opposite trend at 10 DAF (Figures 4f, S6f). GA-synthetic genes,\emph{KS}
(\emph{OsKS1} and \emph{OsKS4} ), \emph{GA20ox}(\emph{OsGA20ox2} and
\emph{OsGA20ox3} ), and \emph{GA3ox}(\emph{OsGA3ox1} and \emph{OsGA3ox2}
), were uniquely expressed in embryo at 5-20 DAF, implying that the
embryo may be main site for GAs synthesis (Xue, Zhang, \& Xue, 2012;
Zhang et al., 2020a). In addition, two GA-deactivating genes, \emph{EUI}
and \emph{OsGA2ox3} , were specifically enriched in endosperm at 5 DAF
and 15-20 DAF, respectively.

Auxin gradually increased until 10-15 DAF in embryo and endosperm, then
decreased thereafter (Figures 4g, S6g). Compared with endosperm, embryo
had higher content at 5-20 DAF. Comparison of gene expression revealed
differential auxin biosynthesis in embryo and endosperm. The step
catalyzed by YUCCAs and TARs occurred in both embryo and endosperm at
5-20 DAF, whereas that catalyzed by TAA1 (\emph{OsTAA1;4} ) occurred
mainly in endosperm at 5 DAF.

ABA content in embryo and endosperm showed two peaks at 15 DAF and 25
DAF in embryo and endosperm (Figures 4h, S6h), in line with the biphasic
pattern of the embryonic development and endorsed the findings of
previous study (Qin, Tang, Pan, \& He, 1990). The expression profiles of
the ABA-synthesis genes in embryo and endosperm also displayed two
peaks. In embryo, \emph{NCED1} , \emph{OsZEP1} and \emph{ABA2} were
highly expressed at 5 DAF, while \emph{NCED3} and \emph{NCED5} at 25
DAF. In endosperm, \emph{NCED1} , \emph{OsZEP1} and \emph{ABA2} were
highly expressed at 5 DAF, while \emph{NCED1} and \emph{OsZEP1} at 20
DAF. In addition, ABA content in endosperm was half of that in embryo.

Isopentenyl transferase (IPT) family members are the rate-limiting
enzymes of cytokinins (CKs) synthesis. \emph{OsIPT4} were specifically
expressed in endosperm, especially at 5-10 DAF (Figures 4i, S6i),
suggesting that CKs have more crucial roles in endosperm than in embryo,
in particular at the early stage to initiate and maintain cell division
or syncytium formation. In embryo, CKs levels gradually increased,
peaked at 10 DAF and then decreased thereafter, consistent with the
expression patterns of CKs-synthetic genes. The CKs in endosperm was
lower than those in embryo at 5-30 DAF and peaked at 5 DAF.

\emph{Minerals}

In embryo, contents of K, Ca, Na, Fe, Mn, Cu and Zn decreased as
development progressed, while those of Mg and P gradually increased
(Figures 5, S9). In endosperm, contents of the nine mineral nutrients
decreased as grain filling progressed, being higher at early stage than
the later stage. This could be attributed to the dilution effect of grain
dry matter, which had a higher rate of accumulation than the minerals
(Wang et al., 2018). Compared with endosperm, contents of all minerals
were high in the embryo.

\textbf{Influence of embryo on the transition of developmental stages of
endosperm}

\emph{A novel method to qualify the effect of embryo on endosperm
development}

As shown in Figure 2, the duration of middle stage in the bottom part of
the NB grain (Figure 2j) was prolonged relative to that of its upper
counterpart (Figure 2i) as well as those of the WT (Figure 2g, h),
indicating a strong influence of the embryo on the bottom endosperm
proximate to it. To quantitively evaluate the embryo effect, we
developed a novel comparison system by comparing the upper and bottom
endosperms of NB, using WT as a reference (Figure 6).

Briefly, this system has three key components: (i) Position effect. The
comparison between the upper (WT\_EnU) and bottom (WT\_EnB) endosperms
of WT 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 (Figure 6a). (ii)
Compound effect. Due to the notched line, movement of nutrients and
signals are severely restricted between the two endosperms, trapping the
influence of the embryo in the bottom endosperm. Comparison between the
bottom part (NB\_EnB) and upper part (NB\_EnU) of the NB grain shows the
compound effect of position and embryo (Figure 6b). (iii) Embryo effect.
Finally, by eliminating the position effect, we can precisely calculate
the influence of embryo on the endosperm via
NB\textsubscript{(EnB/EnU)}/ WT\textsubscript{(EnB/EnU)} (Figure 6c).

To exemplify the working principle of this comparison method, we
selected three metabolites including glutelin, asparagine (Asn), and Zn
to show the influence of embryo (Figure 6). Firstly, taking in account
the position effect and compound effect, the up-regulation of Asn and
down-regulation of glutelin and Zn was observed in both genotypes.
However, when eliminating the effect of position, embryo has a positive
effect on the content of Zn and a negative effect Asn and glutelin,
verifying the influence of embryo on endosperm filling.

\emph{T6P-SnRK1 signaling putatively mediates the crosstalk between
embryo and endosperm}

Using the new comparison method, our results showed that T6P-SnRK1
signaling pathway was active in the endosperm, consistent with that of
the wheat (Mart\selectlanguage{ngerman}ínez-Barajas et al., 2011). As shown in Figure 7, at
early stage of 5 to 10 DAF, embryo had a negative effect on sucrose,
glucose and fructose levels in endosperm during 5-10 DAF, probably due
to its nutrient consumption. The decrease of sucrose content in
endosperm was accompanied by the decline in T6P levels, which in return
inhibited SnRK1 activity (Figure 7a). SnRK1 promoted catabolic activity
in the endosperm, as evidenced by the declining accumulation of starch
and storage proteins (prolamin and glutelin), and the up-regulation of
genes encoding amylase (\emph{AMY3D} ), lipase (\emph{Os01g0651800}
,\emph{Os01g0710700} , and \emph{Os05g0574100} ), and protease
(\emph{OsSAG12} and \emph{OsSCP28} ) in endosperm. Consistently, genes
participating in synthetic process of starch (\emph{OsAGPL4}
,\emph{OsSSSIIIa} , \emph{OsSSIVa} , \emph{OsSSIVb} ,
and\emph{Os02g0807100} ) and storage protein (\emph{OsEnS-115}
,\emph{Prol-14} , \emph{Pro-15} , \emph{Prol13b.20} , and \emph{Glutelin
type-B 2-like} ) were all down-regulated at 5-10 DAF (Figure 7b).

Conversely, at middle stage between 20-25 DAF, T6P-SnRK1 signaling
showed an opposite trend relative to that between 5-10 DAF. Gene
activities of sucrose metabolism (\emph{OsINV1} and \emph{OsNIN6} ) and
T6P synthesis (\emph{OsTPS9} and \emph{Os08g0414700} ) were up-regulated
(Figure 7). SnRK1 activity was inhibited synchronously in endosperm
(Figure 7a). Meanwhile, starch synthetic process was promoted and
catabolic processes were inhibited by SnRK1, as reflected by the
overrepresented starch-synthetic genes (\emph{OsAGPL3} and \emph{ISA1} )
and underrepresented catabolic genes encoding amylase (\emph{AMY3A}
and\emph{AMY3E} ), and protease (\emph{OsAP1} , \emph{Os04g0535200}
,\emph{Os08g0267300} , \emph{Os05g0403000} , \emph{Os04g0330900} ,
and\emph{OsSAG12-1} ) in endosperm (Figure 7b; Table S5).

In addition, this comparison method revealed that the influence of
embryo on IAA was similar with T6P but converse with SnRK1 activity
across the developmental stages, decreasing at 10 DAF while increasing
at 15-25 DAF (Figure 7a). Recently, by modulating T6P content in growing
embryos of garden pea (\emph{Pisum sativum} ), Meitzel et al. (2021)
found that auxin acts downstream of T6P to facilitate seed filling. Our
finding of the synchronously dynamic pattern of IAA and T6P indicates a
similar role of auxin-T6P pathway in grain-filling process of rice.

\textbf{DISCUSSION}

Embryo-endosperm relationship is one of the determining factors
contributing to seed development and subsequently the yield and quality
of cereal grains. However, signals or regulators that coordinate the
developmental processes of the two compartments remain largely unknown.
This study took advantages of the NB mutant to evolve a novel comparison
method for quantifying the influence of embryo on endosperm development,
revealing that the embryo has a dragging effect on the developmental
transition of the endosperm. To our knowledge, it is the first attempt
to directly disclose the evidence of embryo-endosperm interaction during
rice seed development. The findings reveal new aspects of the role of
embryo in the formation process of rice quality, and help to draw an
integrative picture of seed development from the perspective of agronomy
and crop physiology.

\textbf{1. The dragging effect of embryo on endosperm development}

Seed development is an orchestrated progression through a series of
stages, which can be described by two types of `time': the chronological
time and the developmental time defined by the sequence of stages
(Ebisuya \& Briscoe, 2018). There is evidence of bidirectional
interaction between embryo and endosperm throughout development (An et
al., 2020). Notably, two recent studies in Arabidopsis suggest an
independent relationship between the two tissues. By analyzing mutants
with defective endosperm cellularization, O'Neill, Colon, and Jenik
(2019) found that this endosperm process is not required for the onset
of embryo maturation. Using single-fertilization mutants, Xiong, Wang,
and Sun (2021) demonstrated that in the absence of embryo, endosperm
develops the same way as in the wild type, suggesting that endosperm
development is an autonomously programmed process independent of
embryogenesis. These two studies in combination indicate that in terms
of developmental time, the mechanisms controlling endosperm and embryo
development act independently of each other (O'Neill et al., 2019). On
the contrary, in terms of chronological time, the current study show
that duration of endosperm filling in the bottom part of the NB grains
was prolonged by the embryo (Figure 2j), suggesting a substantial
interaction between embryo and endosperm. Similarly, Xiong et al. (2021)
showed that rapid embryo expansion can significantly accelerate
endosperm breakdown, thus shortening the lifespan of the transient
endosperm. Further, it should be noted that cereal seeds like rice have
a persistent endosperm, whereas dicots like Arabidopsis have an
ephemeral endosperm that degrades as the embryo grows. As a result,
whether the independence of endosperm development from embryo in
Arabidopsis is applicable to rice awaits further investigation.

Plants have evolved a variety of timing mechanisms that integrate
chronological time with developmental time to ensure proper development
(O'Neill et al., 2019). For the endosperm, these include internal timers
of molecular oscillators based on hormones or metabolites, and external
timers dependent on environmental signals or emanating from a different
tissue like the embryo. This study reveals a dragging effect of embryo
on endosperm development in chronological time, i.e. extending the
storage accumulating stage whereas delaying the maturation stage. This
finding provides direct evidence for the role of embryo as the external
timer controlling endosperm development. In addition, hormones like GA,
auxins, and ABA were unevenly distributed in rice seed, with the embryo
generally having higher contents at the early and middle stages, as also
reported by Zhang et al. (2020a). Interestingly,
\emph{GA20ox}(\emph{GA20ox2} and \emph{GA20ox3} ), \emph{GA3ox}
(\emph{OsGA3ox1} and\emph{OsGA3ox2} ), \emph{KS} (\emph{OsKS1} and
\emph{OsKS4} ) were predominantly expressed in embryo at 5-20 DAF,
indicating that embryo may be the GA-synthetic site whereas endosperm is
the GA-acting site (Figure 4f). It is well established that
embryo-derived GA modulates the secretion of starch-degrading enzymes
like \selectlanguage{greek}α-\selectlanguage{english}amylase from the aleurone and scutellum upon germination. But it
is still uncertain whether the degradation of starch during seed
development is analogous to the germinating process. Our results imply
that one of the external timers coordinating rice grain development
might be the hormone GA released from endosperm, which needs further
investigation.

For internal timers modifying endosperm development, the T6P-SnRK1
signaling pathway may be the key component. At early stage of 5 to 10
DAF, sucrose in endosperm was lower, probably due to the deprivation by
the growing embryo. In response to the reduced sucrose content, T6P was
decreased simultaneously in endosperm, thus relieving the inhibition of
SnRK1 activity. The increased SnRK1 activities promoted the catabolism
or suppressed the anabolism of starch and proteins, as reflected by the
lower content of starch and prolamins as well as the enhanced gene
activity of amylase, lipase, and protease in endosperm (Figure 7).
Conversely, at middle stage between 20 and 25 DAF, the T6P-SnRK1
signaling showed an opposite trend relative to that between 5 and 10
DAF, indicating it may be involved in the transition of developmental
stages in endosperm (Figure 7). Taken together, the anticorrelation
between T6P and SnRK1 activity opens the possibility that the T6P-SnRK1
pathway may be a master regulator coordinating the communication between
embryo and endosperm during rice grain formation.

\textbf{2. The active role of embryo in grain quality formation}

Grain chalkiness is a highly undesirable quality trait that negatively
affects not only appearance quality but also milling, eating, and
cooking quality. High occurrence of chalky grains has been a major
obstacle for rice producers under the likely scenario of global warming
(Jagadish, Murty, \& Quick, 2015). The physiological and molecular
mechanisms responsible for chalky tissue formation are largely elusive.
Previously, we employed the NB mutant to compare the transcriptome and
proteome profiling of the chalky bottom part and the translucent upper
part of the notched-belly grains, and identified some key pathways
associated with chalkiness occurrence, such as the shifts between C and
N metabolism, and the reduced accumulation of 13 kDa prolamin subunit
(Lin et al., 2017). In addition, we uncovered a substantially negative
influence of embryo on the chemical storages of endosperm (Lin et al.,
2016), highlighting the necessity of discovering the causal factors
behind grain chalkiness from perspective of embryo-endosperm
interaction.

In this study, we successfully applied a novel comparison method to show
the direct effect of embryo on the developmental processes of endosperm.
Using WT as a reference, a comparison of the bottom and upper parts of
NB grains confirmed the findings of our previous work as well as
identified new genes responsible for grain chalkiness formation. In
brief, during the critical period of grain filling (5-20 DAF),
down-regulation of genes encoding prolamin, while up-regulation of genes
related to carbohydrate catabolism and transition metal ion transport
play a key role in chalkiness formation in bottom part of NB grains
(Figure S10a). In addition, the embryo down-regulated genes in endosperm
that relate to cytoskeleton, MCM complex, chromosome segregation, cell
cycle and starch metabolic process, while up-regulated those involved in
lipid transport, biotic stress resistance and starch hydrolysis (Figure
S10b). Notably, the dragging effect of embryo on endosperm provides a
fresh perspective of chalkiness formation. As reported by Morita,
Yonemaru, and Takanashi (2005) and Shi et al. (2017), heat stress caused
rapid proliferation and expansion of the endosperm cells. As a result,
high occurrence of chalky tissue was considered to be associated with
the incomplete accumulation of starch that can not completely fill the
increased endosperm cells. Conversely, our current findings indicate
that the slowdown of endosperm development is another cause for
chalkiness formation. It is thus inferred that a perfect rice grain
without chalky tissue demands a delicate balance between the enlargement
of endosperm size and the accumulation of storage products. Hence, the
timing of transition from cell differentiation to storage accumulation
is critical for the formation of grain chalkiness, and the role of
embryo in this transitional process requires extensive investigation.

\textbf{3. The integrative landscape of rice seed development}

One of the major goals of crop production is to produce more grains with
less time, thus improving resource use efficiency. The duration of each
stage of seed development and the timing of transition between them is
of agronomical significance. As reviewed by Olsen (2020), mechanism
regulating the timing of endosperm cellularization has attracted
attention due to its positive correlation with endosperm and seed size.
Consequently, a precise and integrative landscape of seed development is
necessary and useful for both fundamental and applied studies on the
mechanisms underpinning crop yield and quality. Previously, from
viewpoint of crop physiology, we proposed a practical staging system
with three phases: embryo morphogenesis, endosperm filling, and seed
maturation (An et al., 2020). Here, we update this staging system by
integrating of molecular, physiological, and anatomical evidence in the
current study as well as from the literature. In particular, considering
the dragging effect of embryo on endosperm development at the former
second stage (endosperm filling), we highlight the importance of this
stage as critical for grain filling and quality formation, and thus
subdivide it into two stages: embryo enlargement and endosperm filling.
Accordingly, we paint a holistic and dynamic picture of rice seed
development (Figure 8), and provide a brief description of each stage
and its agronomical relevance as follows.

\textbf{Stage I} : Morphogenesis (0-10 DAF): After double fertilization,
patterning and differentiation occurs simultaneously in the embryo and
endosperm. At the end of this stage, most of morphogenetic events in
embryo have already occurred. Endosperm has finished differentiation,
forming two subregions, the aleurone and starchy endosperm, and starts
storing starch and proteins.

\textbf{Stage II} : Embryo enlargement (10-20 DAF): Embryo greatly grows
to its maximum volume at 20 DAF (Itoh et al., 2005). Starchy endosperm
attains its highest rate of storage accumulation (Fu et al., 2013; Zhu,
Ye, Yang, Peng, \& Zhang, 2011), while the aleurone cells is filled with
aleurone particles and spherosomes at the end of this stage (Yu, Zhou,
Xiong, \& Wang, 2014). This stage witnesses strong interactions between
embryo and endosperm, hence being critical for rice grain filling and
quality.

\textbf{Stage III} : Endosperm filling (20-30 DAF): Embryo becomes
dormant and endosperm continues to accumulate starch and proteins,
reaching its maximum weight at 30 DAF. By the end of this stage, most of
the starchy endosperm and maternal tissues have undergone PCD, losing
their biological activity, while the aleurone and embryo is still alive
(Wu, Liu, Li, \& Liu, 2016b; Wu, Liu, Li, \& Liu, 2016c).

\textbf{Stage IV} : Maturation (30 DAF-maturity): After completion of
reserve accumulation, the embryo becomes tolerant of desiccation, and
undergoes a developmentally programmed dehydration event leading to
dormancy and a quiescent state (Angelovici, Galili, Fernie, \& Fait,
2010; Manfre, LaHatte, Climer, \& Marcotte, 2009). The starchy endosperm
cells die completely upon seed maturation and desiccation. At this
stage, seeds are susceptible to germination under hot and humid
conditions, thus being vulnerable to preharvest sprouting (Du et al.,
2018).

The integrative landscape from Figure 8 has important implications in
crop science and management. It draws distinctive patterns of embryo and
endosperm development, with endosperm ceasing storage accumulation at 30
DAF, ten days after the corresponding timepoint of embryo (20 DAF).
Figure S11 shows that the capacity of embryo to germinate starts at 15
DAF and peaks at 20 DAF for both genotypes of WT and NB. It is thus
inferred that the embryo has the priority of nutrient allocation over
endosperm during seed development. Moreover, it appears that this
asynchrony of embryo and endosperm development is conserved across
modern cultivars. They share a common chronological time, with embryo
developmentally maturing at 20 DAF (Armenta-Medina et al., 2021; Itoh et
al., 2016), while endosperm matures at 30 DAF (Chen et al., 2013; Morita
et al., 2005; Wang et al., 2008; Wei et al., 2017; Wu et al., 2016c; Xu
et al., 2021; Yang, Zhang, Wang, Liu, \& Wang, 2006). Therefore, the
essential period of rice yield and quality formation is the Stages I-III
(Figure 8), from anthesis to 30 DAF. After that, rice seed enters the
stage of desiccation and maturation, lasting for 20 to 40 days with no
marked increase in grain weight. By dividing the 60-day period of grain
filling into two separate months, our previous report showed that only
10 \% of the grain yield was formed after 30 DAF (Xu et al., 2021).
Under the intensive rice-wheat and rice-oilseed rape cropping systems in
the lower reaches of Yangtze River, China, late maturity of the rice
crop causes the late sowing of wheat and oilseed rape, adversely
affecting the seedling growth and subsequently the grain or seed yield
(Bai \& Tao, 2017; Zhang et al., 2020b). Accordingly, we suggest an
agronomical intervention to shorten the duration of the late maturation
stage of rice in order to adapt to the double cropping systems. In
addition, future work should exploit more genotypes to verify if this
asynchrony of embryo and endosperm development in chronological time is
conserved in rice as well as other cereal crops like maize and wheat.

\textbf{CONCLUSIONS}

Despite a number of comprehensive studies on the transcriptome atlas of
seed development with high spatio-temporal resolutions, definite
information about the interaction between the two economically important
tissues of embryo and endosperm is still lacking. In this study, we
successfully applied a novel comparison method based on the NB mutant,
demonstrating a direct effect of embryo on the developmental processes
of endosperm. Integrated analysis of transcriptome and metabolites
identified putative regulatory timers that coordinate the developmental
process between embryo and endosperm. The external timers for endosperm
may be hormones like GA secreted from the embryo, while the internal
timers be the T6P-SnRK1 signaling pathway that mediates carbon
allocation in endosperm. In combination with results from molecular,
physiological, and anatomical investigations, we framed a holistic and
dynamic landscape of rice seed development, and explained its
agronomical significance, in particular for the intensive systems of
rice-wheat or rice-oilseed rape double cropping. In contrast to the
quantum leap in fundamental science of genetic control of seed
development, progress on the applied science like the physiology of
grain filling and quality has apparently lagged behind. The integrative
picture of rice grain formation proposed here is of value to bridge this
knowledge gap, and will facilitate the designing of cogent strategies to
enhance rice yield and quality.

\textbf{ACKNOWLEDGEMENTS}

The research was supported by the National Key R\&D Program, Ministry of
Science and Technology, China {[}2017YFD0300103{]}, the National Natural
Science Foundation of China (31771719), and National High Technology
Research and Development Program of China (2014AA10A605). Rothamsted
Research receives strategic funding from the Biotechnological and
Biological Sciences Research Council of the UK. MP acknowledges funding
from the Designing Future Wheat Strategic Programme (BB/P016855/1).

\textbf{CONFLICT OF INTEREST}

The authors report no declarations of interest.

\textbf{AUTHOR CONTRIBUTIONS}

YT had the main responsibility for data collection and analysis. YT and
LA made the figures. FX, GL, YD and MP revised the manuscript. ZL had
the overall responsibility for experimental design, project management
and manuscript preparation. All authors contributed to the article and
approved the submitted version.

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\textbf{FIGURE LEGENDS}

\textbf{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

\textbf{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\ldots{}, and 60 represent sampling timepoints
(days after fertilization).

\textbf{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.

\textbf{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.

\textbf{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.

\textbf{Figure 6.} \textbf{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.

\textbf{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; \selectlanguage{greek}α-\selectlanguage{english}AMY, \selectlanguage{greek}α-\selectlanguage{english}amylase; \selectlanguage{greek}β-\selectlanguage{english}AMY,
\selectlanguage{greek}β-\selectlanguage{english}Amylase. Red and navy rectangles indicate up- and down-regulation,
respectively.

\textbf{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.



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