Seed Architecture Shapes Embryo Metabolism in
Ljudmilla Borisjuk,aThomas Neuberger,b,cJörg Schwender,dNicolas Heinzel,aStephanie Sunderhaus,e
Johannes Fuchs,a,fJordan O. Hay,dHenning Tschiersch,aHans-Peter Braun,ePeter Denolf,gBart Lambert,g
Peter M. Jakob,f,hand Hardy Rolletscheka,1
aDepartment of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany
bThe Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802
cDepartment of Bioengineering, Pennsylvania State University, University Park, Pennsylvania 16802
dBiology Department, Brookhaven National Laboratory, Upton, New York 11973
eInstitut für Pflanzengenetik, Universität Hannover, 30419 Hannover, Germany
fUniversity of Würzburg, Institute of Experimental Physics 5, 97074 Wuerzburg, Germany
gBayer CropScience, 9052-Zwijnaarde, Belgium
hResearch Center Magnetic Resonance Bavaria, 97074 Wuerzburg, Germany
Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we
demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the
developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration
became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of
storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and
were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the
outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner
cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant high-
light conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase–bypass to seed storage
metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro– versus in
planta–grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We
conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an
efficient use of available light and space.
The shape, size, and architecture of the seed affect many aspects
of a plant’s ecology and evolution (Moles et al., 2005; Gegas
et al., 2010; Muller-Landau, 2010). The size of the seed is of high
relevance for crop production, and seed yield has been and re-
mains the key trait in many breeding programs. Small-seeded
species have evolved to produce large numbers of progeny,
among which only few will complete their life cycle; by contrast,
large-seeded species produce few seeds, but the surviving
seedlings are generally robust enough to ensure that a high
proportion completes its life cycle. Seed development in the
small-seeded species Arabidopsis thaliana has been thoroughly
investigated (Spencer et al., 2007; Niu et al., 2009; DeSmet et al.,
2010; North et al., 2010; Sun et al., 2010; Xiang et al., 2011),
allowing for some of the molecular bases for determining seed
size and pattern formation to be elucidated (Garcia et al., 2003;
Ohto et al., 2005; Ingouff et al., 2006; Adamski et al., 2009). After
fertilization, the growing embryo establishes a bilateral symmetry,
followed by bending and folding of cotyledons due to the physical
restrictions imposed by the testa and the endosperm. It is relevant
to ask whether these morphological changes in themselves have
an effect on the metabolism of the embryo and whether embryo
bending in turn affects local growth conditions. Similarly, posi-
tional/architectural cues may underlie some of the well-established
gradients of gene expression (Belmonte et al., 2013) as well
as those for storage products and metabolite concentrations
(Borisjuk et al., 2005; Rolletschek et al., 2005; Li et al., 2006;
Andriotis et al., 2010). It has recently been possible to demon-
strate that the architecture of the cereal caryopsis induces var-
iation in the severity of these constraints, forcing the endosperm
to adjust to localized concentrations of Suc and oxygen (Melkus
et al., 2011; Rolletschek et al., 2011). This adjustment causes
metabolic heterogeneity within the starchy endosperm and pro-
vides the means to substantially improve the nitrogen and carbon
use efficiency of the caryopsis.
Unlike Arabidopsis, its close relative oilseed rape (Brassica
napus) produces seeds of high economic value. The lipid content
1Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Hardy Rolletschek (rollet@
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved. 1 of 16
of the seed of elite varieties is ;50%, and, consequently, oilseed
rape has become the third most valuable oil crop on a global
basis. The storage metabolism in its seed is therefore of great
interest, particularly in the context of making further improve-
ments in lipid content (Abbadi and Leckband, 2011). The regu-
lation of lipid storage has been reviewed repeatedly (Baud and
Lepiniec, 2009; Weselake et al., 2009; Wallis and Browse 2010),
and there is evidence that the metabolic machinery used by
Arabidopsis is readily transferable to oilseed rape (Niu et al.,
2009). Although most of the genes involved in lipid storage have
been well characterized, the control of metabolic flux in vivo re-
mains obscure. Recent data suggest that lipid assembly exerts
significant control over oil accumulation in oilseed rape (Taylor
et al., 2009; Tang et al., 2012), which, however, contrasts with
other species (Ramli et al., 2009), and the final levels of tri-
acylglycerol seem to be only poorly correlated to the transcrip-
tional activity of the corresponding genes (Troncoso-Ponce et al.,
2011). Neither the spatial regulation of lipid storage nor the ex-
istence of any positional cues imposed by seed architecture have
been explored to date, even though it has been shown that dis-
tinct embryo components (radicle/hypocotyl versus cotyledons) in
both Arabidopsis and oilseed rape accumulate different levels of
lipid at the mature stage (Li et al., 2006).
The absence of the required positional information is in part
attributable to a lack of appropriate analytical methods. How-
ever, recent developments in magnetic resonance imaging (MRI;
Borisjuk et al., 2012) and mass spectrometry have begun to
remove this limitation, thereby allowing for the spatial mapping
of storage lipids (Neuberger et al., 2009; Fuchs et al., 2013) and
their individual components (Horn et al., 2012). In this article, we
designed an array of spatial high-resolution techniques, which
allowed us not only to visualize steep gradients in oil deposition
within the embryo of oilseed rape but also to define their spatial/
temporal relations to those established for the accumulation of
starch and storage proteins, cellular growth, photosynthetic
activity, and metabolite pattern. A high metabolic heterogeneity
became apparent and was further explored by applying tissue-
specific metabolic modeling. Our findings demonstrate how the
embryo is able to make local metabolic adjustments to its en-
vironmental and growing conditions and provide a mechanistic
view on the role of seed architecture for embryo metabolism in
Embryo Topology Analyzed by MRI-Based Modeling
The final seed size (;2 mm) of oilseed rape is established initially
by the growth of the testa and endosperm; later, the embryo
becomes the major site for assimilate storage (Figures 1A and
1B). As the amount of space available for expansion becomes
limited, the embryo is forced to bend and fold (Figures 1A and
1E). To monitor the changes in the shape and volume of the
embryo and its various components we applied noninvasive MRI
(see Methods). At ;20 d after pollination (DAP), the testa, both
cotyledons, and the embryo axis are clearly distinguishable
(Figure 1C). The MRI-based estimates of embryo volume were in
good agreement with weight-based determinations (Figure 1D).
The outer cotyledon expanded more rapidly than either the radicle
or the inner cotyledon, growing to become the largest single
component of the seed (Figure 1D). As development proceeded,
the outer cotyledon expanded to cover both the inner cotyledon
and (partly) the embryo axis, thereby limiting the latter’s access to
light and restricting their space. Such subordination in growth
causes distinct local conditions inside of the seed, possibly
having feedback effects on embryo metabolism in a tissue-
specific manner. We therefore tested the growing embryo for
metabolic heterogeneity at the level of photosynthetic activity,
storage product deposition, component-specific growth rates,
High-Resolution Quantification of the Photosynthetic
Capability of Various Seed Tissues
The photosynthetic energy transfer occurring within the seed
was assessed by the measurement of the linear electron transport
rate (ETR). Both the testa and the outer cotyledon displayed
substantially higher levels of ETR during the main storage
phase compared with the inner embryo region (Figure 2A).
When exposed to close to saturating light levels, the ETR in
the inner cotyledon was only 67% of that obtaining in the
outer cotyledon (Figure 2B) and much higher in seed coat.
This gradient in photosynthetic performance is even fortified in
planta by the gradient in light supply from the outside to the
inside of the seed.
Spatial Profile of Lipid Deposition during
In vivo MRI-based lipid mapping (Neuberger et al., 2009) applied
at distinct developmental stages allowed the distribution of the
lipids within an individual seed to be documented in a three
dimensional format (see Supplemental Movies 1 to 3 online).
This also allowed us to relate structural changes of the embryo
to storage pattern of lipids during development (Figure 3). In the
early stage (Figures 3A and 3E), lipids were recognized first
within the outer region of the endosperm (aleurone) and the
endosperm layer surrounding the embryo, as well as in the
embryo proper. Within the latter, the highest lipid concentration
was in the radicle. Endosperm was the major location for lipid
deposition at this stage. As development continued, a similar
pattern of lipid deposition was retained, although the lipid level
in the embryo increased substantially (Figures 3B and 3F). The
outer cotyledon lipid content was much higher than that in the
inner cotyledon. A pronounced lipid gradient was established
within the outer cotyledon and also within the embryo axis, with
twofold concentration differences within distances of only
;300 µm. During the late storage stage, the lipid level of all of
the embryo components rose (Figures 3C and 3G). Within the
embryo axis, the level achieved in the root apex was higher than
elsewhere, but declining markedly toward the hypocotyl. The
parenchymal tissue within the axis was consistently more lipid
rich than the cells within its central cylinder. The lipid gradient
through the outer cotyledon became less marked, but retained
its higher level than in the inner cotyledon. The outer tissue
2 of 16 The Plant Cell
layers facing the endosperm and testa contained the highest
content of lipid. The fatty acid composition of the lipid fraction
within the specific embryo components sampled at this stage
(obtained from dissected material analyzed by gas chromatog-
raphy) was rather uniform, but differed from that found in the
seed coat (with attached endosperm; see Supplemental Figure 1
online). By the maturation stage (Figures 3D and 3H), the lipid
level throughout the embryo was substantially increased. The
earlier differences between the inner and outer cotyledon with
respect to lipid content had essentially disappeared. Ongoing
lipid accumulation in the cotyledons caused much higher lipid
level than in the hypocotyl/radicle.
Based on above MRI analysis, we followed the dynamics in
the relationship between lipid concentration and volume of the
respective embryo organ during development (Figure 4). It ap-
peared that the increases in lipid level could be correlated with
the expansion in the volume of each organ. When the embryo
organs reached maximal volume (at different time points in the
organs), lipid accumulation in embryo was largely completed.
Starch Deposition Is Spatially Regulated and Correlated
with the Volume of the Embryo
During early development, the increase of starch levels was
correlated with an increase in the volume of each component
(Figure 4). Once the cotyledons had reached their maximum
size, the starch level within them began to decline, indicating its
degradation. This conclusion was reinforced by detailed histo-
logical analysis of seed development (Figures 5B to 5G). Starch
first became visible in the testa, followed by the endosperm and
finally the embryo. Within the embryo, starch deposition began
in the radicle parenchyma, then spread first toward the outer and
finally to the inner cotyledon. During the early to mid storage
phase, starch was mostly present in completely differentiated
and expanded cells, suggesting that starch storage at this time
was associated with cell growth. This pattern corresponds to
that of lipid (Figure 3). Starch degradation followed the same
sequence, so that by the later storage stages, no starch re-
mained in the seed (Figure 5F).
Figure 1. Modeling Embryo Architecture in Oilseed Rape.
(A) The development of embryo size and shape. co, cotyledon; ic, inner cotyledon; oc, outer cotyledon; ra, radicle.
(B) Developmental changes in whole seed (black circle), embryo (white), and endosperm/seed coat (gray) fresh weight (FW); means 6 SD (n = 10) are
(C) A cut-away three-dimensional model of the seed (20 DAP) showing the seed coat (green), outer cotyledon (light green), inner cotyledon (red), and
hypocotyl/radicle (yellow); the volumes of each embryo component were estimated from the model.
(D) Volumes of the various embryo components (in mm3) and whole embryo fresh weight (FW in mg) at various storage stages.
(E) Embryos isolated at distinct storage stages, showing the deformation of the embryo and the folding of the cotyledons (indicated by red arrows).
Trade-Off between Growth and Storage 3 of 16
The Storage of Protein and Lipid Is Colocalized
The same seeds analyzed for their lipid distribution by MRI were
also used to immunohistochemically locate the sites for de-
position of oleosin (Figure 5A; for details, see Supplemental
Figure 2 online), cruciferin (Figures 5H to 5N), and napin (see
Supplemental Figure 3 online). The storage of cruciferin protein
was confined to the embryo and endosperm (Figures 5H to
5N), with the radicle accumulating more heavily than the co-
tyledons during the early/mid storage stage (Figure 5J). A
gradient was established from the outer to the inner cotyledon
(Figure 5K). By the late storage stage, accumulation had
ceased in the embryo, and the gradients had dissipated. Both
the temporal and spatial patterns of cruciferin accumulation
were very similar to those for napin and oleosin. Notably, these
patterns also corresponded to those observed for lipid ana-
lyzed by MRI (Figure 3). Thus, the protein and lipid synthetic
machinery needs to compete for substrate, energy, and cellular
space. A more detailed analysis of the proteome was done
using mass spectrometry (Supplemental Data Set 1 online) as
Metabolite Composition Indicates Component-Specific
Carbohydrate and Energy Metabolism
We next tested whether spatially distinct activities in photosyn-
thesis and storage product deposition are reflected in the steady
state level of central metabolites (see Supplemental Figure 4
Figure 2. Gradients in Seed Photosynthesis as Measured by the Imaging-
PAM Chlorophyll Fluorometer.
(A) Color-coded map of the effective quantum yield of photosystem II
measured at 23 µmol quanta per m2per second, showing the distribution
of photosynthetic capability across the seed.
(B) Rapid light response profiles of the photosynthetic ETR in distinct
regions of the seed. Each data point represents the mean 6 SD (n = 5). ic,
inner cotyledon; oc, outer cotyledon; ra, radicle; sc, seed coat.
Figure 3. Embryo Modeling and MRI-Based Quantitative Imaging of
Lipid Distribution in the Developing Oilseed Rape Seed.
Three-dimensional models of the embryo at the early stage (A), the mid
storage stage (B), the late storage stage (C), and the maturation stage
(D). The dotted line indicates the virtual section shown in (E) to (H), in
which the lipid content (color-coded) is shown. Please note the different
color scales. The corresponding three-dimensional models of lipid dis-
tribution are given in Supplemental Movies 1 to 3 online. cc, central
cylinder; en, endosperm; ic, inner cotyledon; oc, outer cotyledon; ra,
radicle; sc, seed coat.
4 of 16 The Plant Cell
online). The quantitatively major metabolites Suc, hexoses, and
most free amino acids were evenly distributed between the outer
and inner cotyledons and the hypocotyl/radicle. The latter, how-
ever, contained statistically significantly lower levels of glycolytic
intermediates, the maturation-related sugars raffinose, stachyose,
and verbascose, the organic acids isocitrate and oxoglutarate,
pentose-5-phosphates, and the redox equivalents NAD, NADPH,
and NADP and higher levels of ADP, ATP, NADH, g-aminobutyric
acid, Suc-6-phosphate, and trehalose-6-phosphate. Apart from
a few compounds (e.g., citrate and citrulline), the metabolite
contents of the two cotyledons resembled one another, and both
were markedly different from that of the hypocotyl/radicle. The
overall picture was one where similar steady state levels for key
metabolites were maintained across the embryo, while some in-
termediates of carbohydrate and energy metabolism in hypocotyl/
radicle differed substantially from that in the cotyledons.
Component-Specific Metabolic Modeling Based on Flux
The above data provide evidence that a significant metabolic
heterogeneity is established during development. In the next
step, flux balance analysis (FBA) was applied to the data to
predict in vivo metabolic fluxes at the main storage phase in the
hypocotyl/radicle, inner cotyledon, and outer cotyledon (models
A, B, and C, respectively). Modeling constraints and predicted flux
values (variability intervals) for each tissue specific model are
given in Table 1 (additional information is given in Supplemental
Data Sets 2, 3 and 4 online). The models predicted a marginal
improvement in carbon conversion efficiency (CCE) due to light
usage, although the contribution of photosynthesis to seed me-
tabolism was sizeable when a comparison was made between
the rate of photolysis and the demand for NADPH. Only in the
inner cotyledon was the effect of photosynthesis negligible
(i.e., the component was growing heterotrophically). In all three
components, ribulose-1,5-bis-phosphate carboxylase/oxygenase
(Rubisco) was predicted to have been essentially inactive, pre-
sumably because it is only when the light level exceeds a certain
threshold that the light-dependent improvement of CCE depends
on an increasing flux through Rubisco (Hay and Schwender,
2011b). Here, the predictions were based on a light intensity of
400 µmol quanta per m2per second (as in the growth chamber);
had the light level been increased to full sunlight, then the con-
tribution of Rubisco to seed metabolism was predicted to have
become substantial for both the outer cotyledon and the hypo-
cotyl/radicle (Figure 6A). With increasing light, CCE can be ex-
pected to rise and the net CO2emission to fall, reaching one in the
outer cotyledon at ;1930 µmol quanta per m2per second. This
extrapolation suggests that under high light conditions, the Ru-
bisco bypass (Schwender et al., 2004) substantially contributes to
seed storage metabolism in oilseed rape, yet only in the outer
cotyledon and the hypocotyl/radicle.
The effect on the plastids on the availability of NADPH/
ferredoxin was assessed in all three components (Figure 6B). In
the outer cotyledon and hypocotyl/radicle, ;58% of the plasti-
dic reductant was derived from photosynthetic electron trans-
port compared with just 3% in the more heavily shaded inner
cotyledon (Table 1). The analysis indicated that photosynthesis
contributes essentially nothing to synthesis in the inner cotyle-
don. With respect to plastidic ATP, ;80% was derived from
photosynthesis (thylakoid H+transporting ATPase, #92) in the
outer cotyledon and hypocotyl/radicle, compared with just 4%
in the inner cotyledon (Table 1).
Using a lumped network projection and normalizing metabolic
fluxes relative to the sugar uptake rate (=100%), the flux distri-
bution within the central carbon metabolism was compared
between the various embryo components (Figure 6C). This
showed that the relative flux associated with the majority of the
reactions was comparable across the three components.
However, clear differences were noted for the tricarboxylic acid
(TCA) cycle, where the inner cotyledon had a rather higher flux
than was present in either the outer cotyledon or in the radicle/
hypocotyl (Figure 6C). In addition, mitochondrial ATP synthesis
(H+-transporting two-sector ATPase, #91) was higher in the in-
ner cotyledon than in either the outer cotyledon or the hypo-
cotyl/radicle (see Supplemental Data Set 3 online).
Lipid Deposition and Photosynthetic Electron Transport
in the Embryo Both Respond to Growing Conditions
To test how embryo architecture and light supply affect the
tissue specificity of metabolism, we manipulated growth con-
ditions in two ways. First, developing embryos were isolated and
cultured in vitro. As the growing embryo experienced no space
restrictions imposed by the endosperm/testa, the cotyledons
did not fold/deform (Figure 7A), allowing each to receive a uni-
form level of light. Under these circumstances, no lipid gradient
was established in the embryo nor was there any statistically
significant variation in ETR across the cotyledons (Figures 7B and
7C; see Supplemental Movie 5 online). Second, the developing
silique in planta was covered with a nontransparent material
Figure 4. Temporal Pattern of Lipid and Starch Accumulation in Various
Embryo Components in Relation to Their Volume.
Top panels: lipid concentration versus component volume; bottom
panels: starch concentration versus component volume. Error bars in-
dicate SD (n = 6). The MRI model used for calculation of lipid/volume
ratios at mid storage stage is given in Supplemental Movie 4 online. DW,
Trade-Off between Growth and Storage5 of 16
(aluminum foil) for 10 d. As light was excluded from reaching the
embryo, there could be no photosynthetic contribution to its
growth. Instead, there was a component-specific repression of
lipid storage (31% in the outer cotyledon, 18% in inner cotyledon,
and 0% in the radicle/hypocotyl; see Supplemental Figure 5
online). Analysis of the fatty acid composition of the lipid fraction
showed that the contribution of unsaturated fatty acids (linoleic
and a-linolenic acid) was depressed in the absence of light. The
experiment demonstrated that photosynthesis in the embryo has
the most pronounced effect on the outer cotyledon.
The Combined Response to Light of the Embryo Proteome
An analysis of the total embryo proteome identified 96 spots as
differing between the illuminated and the nonilluminated de-
veloping seed (see Supplemental Data Set 5 online). Mass
spectrometry was able to identify 13 distinct proteins belonging
to eight functional categories that were of increased abundance
under light; among these were several storage proteins. Under
the nonlit conditions, 77 proteins were produced in increased
abundance; most of these belong to the categories “carbohy-
drate and amino acid metabolism” and “genetic information
processing.” Several enzymes associated with glycolysis, fatty acid
oxidation, and amino acid metabolism featured among the 77
identified proteins (Figure 8), and the conversion between ATP and
ADP via nucleoside diphosphate kinase was of greater importance
in the absence of light. The overall pattern suggested that the
absence of light induced the glycolytic pathway and generated
a shift in amino acid metabolism, while its presence favored the
transcriptional/translational activation of storage metabolism.
The illuminated and the nonilluminated developing seed were
also compared at the steady state metabolite level (Figure 8; see
also Supplemental Data Set 6 online). Liquid chromatography–
mass spectrometry–based analysis demonstrated that sugars
Figure 5. Colocalization of Oleosin, Cruciferin, and Starch in the De-
veloping Oilseed Rape Seed.
(A) Schematic representation of oleosin distribution (for details, see
Supplemental Figure 2 online).
(B) to (F) Starch deposition (iodine staining).
(B) Starch grains are visible within the seed coat and endosperm during
early storage stage.
(C) Starch is no longer detectable in the seed coat during the later
(D) A cross section through the seed shows the presence of starch in the
seed coat, endosperm, and embryo, visible as a gradient from the radicle
toward the cotyledon. The outer cotyledon is more heavily stained than
the inner one.
(E) A gradient in starch content within the outer cotyledon (cf. with the
protein gradient shown in [K]).
(F) A cross section through the mature seed shows the complete ab-
sence of starch.
(G) Schematic representation of starch distribution.
(H) to (M) Cruciferin deposition (immunostaining).
(H) A cross-section through the seed coat and endosperm during the
early storage stage shows no cruciferin to be present;
(I) The mid storage stage endosperm contains plenty of cruciferin.
(J) A cross section of the young seed demonstrates the presence of
cruciferin in the embryo and cotyledons. A gradient in concentration is
visible in both radicle and cotyledons.
(K) A cross section through the cotyledons during the mid storage stage
shows a cruciferin gradient within the outer cotyledon. The storage va-
cuoles in the abaxial region are full of protein (double arrow left), those
closer to the seed’s interior are less filled, while those in inner cotyledon
are empty (double arrow right).
(L) Cortical tissue of radicle showing cells completely full of cruciferin.
(M) The cotyledonary vascular tissue is relatively free of cruciferin.
(N) Schematic representation of cruciferin distribution.
al, aleurone layer; cc, central cylinder; en, endosperm; ic, inner cotyle-
don; oc, outer cotyledon; ra, radicle; sc, seed coat; vs, vascular tissue.
6 of 16The Plant Cell
and hexose phosphates persisted at much lower levels under lit
conditions and that the content of most of the free amino acids
and of all of the free fatty acids was drastically reduced by the
presence of light. We further noticed differences in the levels of
trehalose-6-phosphate (known to regulate carbon partitioning;
Ponnu et al., 2011), ATP, and oxygen under lit versus nonlit
conditions. Other energy metabolites, such as ADP and pyro-
phosphate, were maintained at comparable levels. The overall
picture is one where metabolite levels (so presumably metabolic
pathway activity) differed substantially between the light re-
gimes. The reduced levels of sugars, free amino acids, and free
fatty acids might indicate a higher turnover rate for enhanced
accumulation of storage products under lit conditions.
The application of FBA to the outer cotyledon indicated a ris-
ing flux of glycolytic and other enzymes, including Suc synthase,
phosphofructokinase, Fru-biphosphate aldolase, triosephosphate
isomerase, enolase, and malic enzyme (reactions #30, #63,
#25, #24, #536, and #49 in Supplemental Data Set 4 online) in
response to a decrease in light availability. Correspondingly,
our proteome data set confirmed an elevated abundance for
each of these enzymes under nonlit conditions (Figure 8).
Concentration changes were also noted for the relevant
Table 1. Model Constraints and Model Predicted Exchange Fluxes and Other Rates for Various Organs of Developing Oilseed Rape Embryos in Planta
Sub-Model (In Planta, 32 DAF)
Flux value constraints
Growth rate (Biomass_exch)a
Photon uptake flux (Ph_tm_exch)
Nitrate uptake (NO3_ap_exch)
Ammonia uptake (NH4_ap_exch)
mg DW h21
Cell wall (glucose polymer)
Fatty acid composition, mid stageb
g/ 100 g fatty acids
g/ 100 g fatty acids
g/ 100 g fatty acids
g/ 100 g fatty acids
g/ 100 g fatty acids
g/ 100 g fatty acids
g/ 100 g fatty acids
Model predicted exchange fluxes and flux ratiosc
Relative Rubisco fluxd
CCE (no light)
Relative contribution of photosynthetic electron transport
to demands of plastidic syntheses via NADPH,
Relative contribution of light driven ATP production to
plastidic ATP consumption
78.6% 4.1%80.6% NA
aReaction names, Supplemental Data Set 3.
bBased on biomass composition, model specific equations for the reactions “TAGsynth_c”, “Lipase_c” and “ Biomasssynth_nc, ap, c, p” are specified
in Suppl. Table 1.
cNegative flux values denote influx.
das defined in Schwender et al. (2004)
ecarbon conversion efficiency
DAP, days after pollination; DW, dry weight; NA, not applicable.
Trade-Off between Growth and Storage7 of 16
Figure 6. Simulation of Component-Specific Metabolism in the Oilseed Rape Embryo.
(A) Relative flux through Rubisco.
(B) Plastidial reducing equivalents (NADP and ferredoxin, redFD) redox balance in the outer cotyledon, inner cotyledon, and radicle (from top to bottom)
at a simulated illumination level of 400 µmol quanta m22s21. Each component is indicated by a specific color. All influxes and effluxes are shown, as
8 of 16 The Plant Cell
metabolic intermediates. Assuming relative enzyme abun-
dance to be usable as a proxy for changes in flux (Sweetlove
and Ratcliffe, 2011), the experimental data validated the pre-
dictions derived from the FBA approach.
Comparison of Embryo Growth in Planta versus in Vitro
We performed comparative analysis of in planta– versus in vitro–
grown embryos (;30 DAP) at the level of biomass composition,
proteome, and metabolome. The in vitro–cultivated embryos
showed fully opened, equally sized cotyledons and continued to
expand, reaching higher biomass than the in planta–grown ones
and also accumulated more starch (see Supplemental Figure 6
online). At the metabolite level, in vitro–grown embryos showed
higher levels of most sugars and free amino acids, as well as
various changes in glycolytic and TCA cycle intermediates in
comparison to in planta–grown embryos (see Supplemental
Figure 6 online). At the proteome level, we found significantly
decreased abundance of proteins belonging to storage and
photosynthesis under in vitro conditions but increased abun-
dance of proteins involved in stress response and glycolysis
(see Supplemental Data Set 7 online).
Owing to their sessile lifestyle, plants have developed various
adaptations to cope with a changing environment (light, stress,
and physical space). Our study demonstrates that the tiny
rapeseed (Brassica napus) embryo exercises local metabolic
adjustments already during its development inside of the seed.
The spatially resolved analysis of gene expression in seeds can
provide essential cues on cellular metabolic functions (Spencer
et al., 2007; Belmonte et al., 2013) but only limited information
on metabolic activities in vivo (Fernie and Stitt, 2012). Getting
the whole picture therefore requires one to follow metabolism at
the level of metabolic intermediates, storage products, and
fluxes, all if possible at a high spatial resolution and in vivo. Here,
we analyzed embryo metabolism at a high level of spatial res-
olution by applying noninvasive lipid mapping and various other
techniques. The outcome served as input to a tissue-specific
metabolic modeling approach, predicting how the embryo
components adjust to local growth conditions inside the living
seed. We argue that the folding of cotyledons, as observed in
planta, causes a tremendous metabolic heterogeneity in the
developing embryo of oilseed rape.
A Mechanistic View on the Timing and Causes of Metabolic
Heterogeneity in the Oilseed Rape Embryo
The growing embryo has to adjust its shape, size, and metab-
olism to the environment afforded it by the mother plant, which
involves restrictions in physical space and in the amount of in-
cident light. At the early stage of its development, the embryo is
surrounded by a liquid endosperm in which the necessary
sugars and amino acids are homogeneously distributed (Melkus
Figure 6. (continued).
derived from flux values of all reactions that share NADPH_p and redFD_tm. To assess the contribution to plastidial synthetic processes of photo-
synthetic electron transport, the principal electron sources and sinks were first defined and their combined electron flows derived. The identified
producers were photosynthetic electron transport (photosystem I, #461) and various plastidial NADP-dependent oxidoreductases (Glu synthase, #45;
Glu dehydrogenase, #46; malate dehydrogenase, #48; malic enzyme, #49; glyceraldehyde-3-phosphate dehydrogenase, #59; Glc-6-phosphate de-
hydrogenase, #477; phosphogluconate dehydrogenase, #479; isocitrate dehydrogenase, #533).
(C) Metabolic fluxes within each embryo component (from top to bottom: outer cotyledon, inner cotyledon, and radicle/hypocotyl) scaled on the basis of
its sugar uptake rate (=100%). Arrowheads denote the direction of net flux. Dashed arrows represent the summed fluxes into protein (P) or tri-
acylglycerol (T). For details, see Supplemental Data Set 3 online (table “B normalized fluxes”).
Figure 7. Lipid Distribution and Photosynthetic Performance in the in Vitro–Grown Embryo.
(A) Embryo morphology at 32 DAP; inset shows in planta–grown embryo (left) and its lipid distribution (right) at the same developmental stage.
(B) Lipid distribution, as visualized by MRI; the corresponding three-dimensional model is given in Supplemental Movie 5 online.
(C) The light response of the photosynthetic ETR in the cotyledons. Data represent means 6 SD (n = 5).
Trade-Off between Growth and Storage 9 of 16
et al., 2009). As soon as space restrictions induce the bending
of the embryo, the cotyledons grow unequally and gradients
of various storage products are generated. The folding of the
cotyledons results in the inner one becoming ever more en-
closed by and thus controlled by the outer one. This necessarily
includes that the outer embryo regions receive more light en-
ergy, which can be transmitted toward growth and storage
metabolism. The outer cotyledon grew more rapidly, driving
tissue differentiation and thereby forming gradients of starch,
protein, and lipid (Figures 3B and 3C). Both absolute flux rates
Figure 8. Proteomic and Metabolomic Response of the Oilseed Rape Embryo to Illumination.
Changes in protein abundance are indicated in blue (upregulated under nonlit conditions) and red (upregulated under lit conditions). Steady state
metabolite levels are given as means 6 SE (n = 5). Asterisks indicate statistically significant (P < 0.05) differences according to a t test. Raw proteomic
and metabolomic data are given in Supplemental Data Sets 5 and 6 online. DHAP, dihydroxyacetone-phosphate; FW, fresh weight; PGA, phospho-
glycerate; PEP, phosphoenolpyruvate.
10 of 16 The Plant Cell
and relative flux distribution differed between the embryo com-
ponents (Figure 6), which eventually caused distinct contributions
of the embryo components to final seed biomass, oil storage, etc.
Space Use Efficiency as a Strategy for Embryogenesis
Starch synthesis and degradation are contemporaneous in the
embryo (Da Silva et al., 1997), but here were shown to be spa-
tially separated (Figure 5). Once growth has ceased, starch
levels visibly declined at the same time as the accumulation of
lipid and protein continued. The significance of transient starch
storage in oilseeds is controversial. Starch has a low energy
density (i.e., it requires more space per unit energy than does
either lipid or protein). However, since the accumulation of as-
similate in the form of starch is energetically highly efficient
(Schwender, 2008), it is unsurprising to find its accumulation is
associated with the rapidly growing young embryo (Figure 4).
The antisense repression of starch synthesis in the oilseed rape
embryo inhibits Suc import, glycolysis, and respiration and acts
as a brake on growth (Vigeolas et al., 2004). Starch synthesis
thus can be considered as representing a mechanism for cre-
ating a sink (Andriotis et al., 2010) during early embryogenesis,
but when space becomes limiting, it is better for the embryo to
accumulate lipid rather than starch. Once the oilseed embryo
stopped its growth, its starch level declined (Figure 4). This
switch was reached at differing times in the various components
assayed. Under in vitro conditions, the spatially unrestricted
growth of the embryo was associated with a continuous accu-
mulation of starch, resulting in a lesser calorific value biomass
than represented by the in planta–grown seed. The strategy of
redirecting carbon from starch into lipid pushes the embryo
toward a more efficient use of space and ultimately increases
the energy density of the seed. The same strategy is currently
applied in the engineering of plants as a feedstock for bioenergy
production (Ohlrogge et al., 2009; Sanjaya et al., 2011).
Metabolic Modeling Highlights the Tissue Specificity of the
Central Metabolism and Predicts Local Flux Adjustments
Integrating experimental data sets with metabolic models is
necessary to obtain a predictive understanding of metabolism
(Sweetlove and Ratcliffe, 2011; Fernie and Stitt, 2012). Here,
high-resolution measurements of in planta developing embryos
were combined with FBA based on the bna572 metabolic model
for cultured oilseed rape embryos (Hay and Schwender, 2011a,
2011b). The predicted contribution of photosynthesis to growth
and synthesis markedly differed between the outer and the inner
cotyledon (Table 1, Figure 6B). These changes reflected the
embryo’s adjustment to the local level of incident light and the
dynamics of ATP/NADPH provision. The light-induced stimula-
tion of lipid storage is well known (Ruuska et al., 2004; Goffman
et al., 2005; Li et al., 2006). Here, we demonstrate how this
translates into gradients in oil deposition inside the developing
seed and highlight the necessary metabolic adjustments that the
embryo makes at a high level of precision up to the cellular level.
The in planta models also predicted that Rubisco is essen-
tially inactive in all three components. The lack of Rubisco flux
relates to a metabolic phase formerly described to be specific
for low-light conditions in embryo cultures (Hay and Schwender,
2011b). In simulations of bna572 that raise the photon flux from
zero to higher levels, the seed carbon balance initially improves
only due to a reduction in decarboxylation reactions like iso-
citrate dehydrogenase. Only with rising of the light flux above
a threshold value, Rubisco flux is predicted to increase and to
improve the carbon balance by refixing CO2(Hay and Schwender,
2011b). In phase one, there is a negative relationship between
photosynthetic activity and TCA cycle activity, which has been
corroborated by comparing flux distributions derived by
metabolic flux analysis for developing embryos of rapeseed and
soy (Glycine max, light grown) and nonphotosynthetic embryos
of sunflower (Helianthus annuus) and maize (Zea mays) (O’Grady
et al., 2012). Similarly, the flux map in Figure 6C shows that the
much more shaded inner cotyledon has a substantially higher
TCA cycle activity than the photosynthetically active outer coty-
ledon. This corresponds to the general view on TCA cycle activity
under the light/dark shift (Lee et al., 2010; Nunes-Nesi et al., 2013).
Given the experimental evidence that the bypassing of the
upper reactions of glycolysis by Rubisco is an active process in
the cultured oilseed rape embryo (Schwender et al., 2004), the
predicted inactivity of Rubisco in planta was a surprising out-
come. The difference between cultured and our in planta
growing embryos is mainly due to the difference in light levels
(see Supplemental Data Set 2 Flux Appendix online). Extrapo-
lation of the in planta models to higher light levels suggested
that in both outer cotyledon and hypocotyl/radicle the Rubisco
becomes active at incident light beyond ;750 µmol quanta per
m2per s (Figure 6A). This defines the tissues and conditions for
the contribution of the Rubisco bypass in nature. We conclude
that under the high light intensities found in the field, Rubisco is
expected to substantially contribute to lipid synthesis and the
carbon economy in the outer cotyledon and the hypocotyl/
radicle, but likely not for the inner cotyledon.
Certain cofactors have recently been shown to be related to
storage capacity for both lipids and proteins (Hayden et al., 2011),
reflecting their higher biosynthetic energy requirements compared
with starch (Schwender, 2008). As a result of being shaded, the
inner cotyledon grows heterotrophically, while the growth of the
outer one is photoheterotrophic. This is in contrast with the in vitro
situation (Figure 7) where both cotyledons receive the same light
and achieve similar photosynthetic ETR and similar oil content/
distribution. Another prediction of the FBA model was that the
outer cotyledon switches from net oxygen uptake to release at
a light intensity above ;920 µmol quanta m22s21(while the inner
cotyledon remains as a net oxygen consumer irrespective of the
incident light level). Thus, the embryo’s photosynthetic activity
has a considerable effect on the seed’s oxygen balance, in par-
ticular, helping it to avoid the onset of hypoxia, which is inimical
to seed metabolism (Tschiersch et al., 2011; P. Verboven,
E. Herremans, L. Borisjuk, Verboeven et al., 2013).
Advantages of in Planta– versus in Vitro–Based Metabolic
To get estimates (predictions) on metabolic activities of the em-
bryo components in planta, we here applied FBA, while previous
Trade-Off between Growth and Storage 11 of 16
modeling attempts largely used13C-metabolic flux analysis (13C-
MFA). In general,13C-MFA is superior in both quantitative and
diagnostic cases (Schwender, 2008) and has provided valuable
quantitative information (e.g., on the effect of nutrient source or
light supply on embryo metabolism) (Junker et al., 2007; Allen
et al., 2009; Allen and Young, 2013). However,13C-MFA essen-
tially relies on in vitro cultivation systems. Thus, its validity de-
pends on how exact it can resemble the in planta situation. For
nonphotosynthetic seeds/embryos, such as maize and sunflower,
culture conditions might in general be closer to in planta since
they are cultivated under continuous dark, and only minor com-
positional differences between in planta versus in vitro are re-
ported (Alonso et al., 2007, 2010, 2011). For green seeds/
embryos, the situation is more complicated as they have to grow
under continuous light. The growth rate is often faster and the
embryos accumulate more starch (Arabidopsis; Lonien and
Schwender, 2009). This was also evident here for oilseed rape,
and we additionally demonstrated how this reflects at the pro-
teome and metabolite level (see Supplemental Data Set 7 and
Supplemental Figure 6 online). Under in vitro conditions, where
the developing cotyledons unfold and receive equal amounts of
light, they also showed a uniform pattern of growth, photosyn-
thesis, and lipid accumulation (Figure 7). From that we conclude
that in vitro–based modeling is less suited to uncover metabolic
heterogeneity of oilseeds and/or to quantify metabolic rearrange-
ments due to developmentally changing seed architecture as
occurring in planta.
In this study, we defined three models to differentiate between
three embryo components, differing in their biomass composi-
tion, growth rate, etc. It should be noted that for in planta
growing embryos, we also reported marked spatial gradients
within the components. Thus, for interpretation of the flux dis-
tributions, one should keep in mind that the three submodels of
embryo metabolism represent a coarse spatial resolution. Yet,
we deem the approach to be a valid approximation that can be
refined in the future, in particular based on the MRI-based
technology presented here.
The FBA modeling framework accounts for metabolic pro-
cesses related to the biosynthesis of cellular biomass synthesis
but does not predict cellular maintenance functions. These
metabolic expenses consume energy cofactors and therefore
are represented as a generic ATP hydrolyzing reaction
(ATPdrain). As it was impossible to accurately measure carbon
balances for the various embryo components in planta, we had
to estimate the ATPdrain flux based on the procedures detailed
earlier (Hay and Schwender, 2011a). The ATPdrain estimate
applied in our FBA is a rather conservative estimate of the size of
this flux. Even twofold increases in ATPdrain in each model did
not affect our major conclusions.
Topology of Lipid Storage and Implications for
Increasing both seed lipid content and quality are important
breeding goals in oilseed rape. The application of the MRI-based
lipid imaging tool has allowed for the topological study of lipid
deposition in a small seed to a resolution of ;30 µm isotropic,
thereby enabling the definition of both “where” and “when” lipids
accumulate. The identification of regions of the embryo that
differ in lipid content provides opportunities to uncover the
transcription factors responsible for the initiation of lipid storage.
Appropriate analytical methods based on laser microdissection
applicable to oilseed rape have recently been established
(Schiebold et al., 2011). Our data have also shown that the rate
of lipid synthesis can vary severalfold between adjacent tissues/
components and depends heavily on the local environment. To
what extent this behavior requires differential gene expression
remains to be established. In this context, however, it may be
relevant that temporal variability in gene expression in the Arab-
idopsis embryo is much higher than its spatial variability
(Spencer et al., 2007), so that the variability shown by the oil-
seed rape embryo may be in part at least governed by the
posttranscriptional regulation of in vivo fluxes. A candidate
mechanism is phosphorylation, and notably most of glycolytic
and TCA cycle enzyme proteins in rapeseed are phosphorylated
(Meyer et al., 2012). The broad scale significance of protein
phosphorylation for central metabolism was recently demon-
strated (Oliveira et al., 2012).
Lipid deposition starts in the endosperm and radicle early
during embryogenesis. During the mid storage stage, lipid gra-
dients are established both within and between the cotyledons
(Figure 3). By maturity, the cotyledons have become uniformly
lipid rich, while the lipid content of the hypocotyl/radicle remains
low, as it does also in other species (Borisjuk et al., 2005;
Neuberger et al., 2009; Horn et al., 2012). A possible approach
to increasing the lipid yield of oilseed rape would therefore be to
upregulate lipid synthesis in the hypocotyl/radicle so as to reach
a level equivalent to that achieved in the cotyledons. Based
simply on the volume occupied by the hypocotyl/radicle, one
can estimate that this would increase the total lipid yield of the
crop by ;8%. Whether increasing the lipid content of this em-
bryo component is even feasible, and if so whether there would
be any negative side effects on maturation/germination remains
to be seen (Shen et al., 2010).
In contrast with what has been concluded previously from
whole-embryo analyses (Murphy and Cummins, 1989), we here
demonstrated that lipid and protein storage occur simulta-
neously. This spatial and temporal overlap appears to be part of
tissue differentiation and represents the basis for competition
among the corresponding biosynthetic pathways for the same
substrates, energy, and physical space. In mature seeds, lipid
and protein content are generally negatively related (Grami et al.,
1977), which most likely reflects this competition. While the re-
direction of substrates from lipid to protein synthesis appears to
be feasible (Chen et al., 2009), the opposite has proven difficult
to engineer to date (Abbadi and Leckband, 2011). Rather, se-
lection for the summed content of protein and lipid has been
shown to be more effective in raising lipid content than has
selection based on just lipid content alone (Grami et al., 1977).
Since achieving any increase in lipid content appears to be
challenging, other strategies may come into play. The metabolic
models predict that the outer cotyledon of oilseed rape is par-
ticularly efficient in using light energy for lipid synthesis. Thus,
increasing the surface area of the seed should increase the
amount of light captured by the embryo and, therefore, the
amount of cofactors/assimilates produced by the embryo
12 of 16The Plant Cell
photosynthesis. It might therefore be advantageous to manip-
ulate the shape of the seed to increase its surface area; this
implies selection against spherical seed. We believe that ex-
ploring the role of seed architecture in storage metabolism could
open new avenues for crop improvement.
Plants of oilseed rape (Brassica napus var HS144B) were grown in
a phytochamber at 18°C with 16 h of light (400 µmol quanta m22s21) and
a relative air humidity of 60%. At the time of flowering, plants were tagged
for determination of developmental stages (DAP). Embryos were isolated
at distinct stages, dissected, and instantly frozen.
All MRI data sets were acquired on a 17.6 tesla Avance III (Bruker) wide-
bore system using an absolute quantification method described else-
where (Neuberger et al., 2009). In brief, four experiments with seeds of
different developmental stages were conducted. The seed was wrapped
in Teflon tape to prevent moisture loss and inserted into a home built
3-mm ID solenoid coil. Before the imaging sequence, a global T1 mea-
surement of the lipid signal was conducted to determine the minimum
repetition time (TR) necessary to avoid for T1 correction during the
quantification process.Asaresultofthismeasurement (T1;700ms;data
not shown), the TR was chosen to be at least ;3*T1 or longer. A standard
multi-slice multi-echo spin echo (MSME) sequence with a CHESS sup-
pression module on the water resonance was applied to acquire high-
resolution lipid images of the seed. An in-plane resolution of up to 20 µm
isotropic and a slice thickness of 105 µm in the seed of the late de-
velopmental stage could be achieved (experimental time 7.5 h). Seeds in
the other developmental stages had slightly lower resolutions to reduce
the experimental time as care had to be taken that no seed shrinkage due
to degeneration occurred (shrinkage of the seed would have resulted in
Either six or eight echoes with a maximum echo time of up to 47 ms
were acquired in the MSME sequence. Apparent T2 fitting on a pixel-by-
pixel basis was conducted using MATLAB (The Mathworks). As TR was
chosentoberelatively long,aT1correction wasnotapplied. Furthermore,
due to the uniform B1 field of the used solenoid coil (data not shown),
a correction for B1 inhomogeneity was not necessary. To generate the
volumes of the different organs in the seed, the first image from the echo
train of each slice was imported into AMIRA (Mercury) and segmentation
was conducted. The average signal in each organ could be calculated
according to Neuberger et al. (2009). This result and the use of a cali-
bration curve shown by Neuberger et al. (2009) enabled a pixel-by-pixel
determination of the absolute lipid content within the seed.
For the measurement of the in vitro culture, the embryo was placed in
a D2O-filled NMR tube with an inner diameter of 4 mm. The tube was
inserted into an in-house-built saddle coil with an inner diameter of 5 mm
and measured using a three-dimensional MSME method (echo time, 4.9
ms; TR, 2 s) with solvent suppression. The achieved isotropic resolution
was 89 µm during the experimental time of 1.75 h. Reconstruction and
visualization was performed using in-house Java-based software.
In Vitro Culture of Oilseed Rape Embryos
Intact embryos were isolated at 20 DAP and kept in liquid culture for 10
dunderphotoheterotrophy (50µmol quantam22s21)at20°Cwithorganic
nitrogen sources according to previous protocols (Schwender et al.,
Chlorophyll Fluorescence Imaging and Light Transmission
Images of chlorophyll fluorescence parameters were obtained using an
Imaging-PAM chlorophyll fluorometer as detailed earlier (Borisjuk et al.,
II can be interpreted as a map of the relative rate of photosynthetic
electron transport (ETR) at identical light supply across the seed tissue
section. The obtained data were also used to create rapid light response
curves. Measurements of light absorbance of pod wall, seed coat, and
outer cotyledon were done using a photometer (LI-250; LICOR). The
effective ETR of embryo organs was calculated based on the light
transmitted to the surface of the individual organ, the organ-specific light
response curve (Figure 2B), and the organ-specific surface area (derived
from the NMR-based embryo models in Figure 3).
Experimental Treatment of Plants
To study the effect of lit versus nonlit conditions on embryo growth,
developing pods were covered by black paper at 10 DAP. After 20 d, the
embryos were isolated and weighed, followed by proteome and me-
tabolite analysis. In some experiments, isolated embryos were dissected
into various organs before analysis.
Freeze-dried embryo material was used for biochemical analysis.
Metabolic intermediates were extracted and analyzed using liquid
chromatography–mass spectrometry as detailed by Rolletschek et al.
(2011). Starch content was assessed spectrophotometrically in the in-
soluble residue following extraction. Total lipid was quantified by NMR
(MQ-60; Bruker) (Borisjuk et al., 2011). The fatty acid composition of total
lipids and free fatty acids were extracted and measured by gas chroma-
tography as described by Borisjuk et al. (2005). Oxygen concentration in-
side the seed was measured using microsensors (Rolletschek et al., 2011).
Proteome analysis of embryo material was analyzed as outlined in the
Supplemental Data Set 1 Proteome Appendix online.
Histochemical techniques applied to seeds as well as immunostaining
were performed as described (Radchuk et al., 2012). Immunolocalization
etal., 2008), antioleosin (20-kD class; Agrisera), andantinapin(Tiedemann
et al., 2008) polyclonal antibody.
FBA was based on bna572, a model for cultured oilseed rape embryos
(Hay and Schwender, 2011a, 2011b). The model was simulated in
a photoheterotrophic mode with organic nitrogen sources available (Hay
and Schwender, 2011a). Three submodels were derived for hypocotyl/
radicle, inner cotyledon, and outer cotyledon, designated as models A, B,
and C, respectively. These differed by constraints derived from organ-
specific in planta physiological data specific for 30 DAP. An overview on
submodel-specific constraints is given in workbook C of Supplemental
Data Set 3 online. Biomass composition, growth rate, photosynthetic
ETR, and light supply were determined at 30 DAP for each of the three
organs (for details, see Supplemental Data Set 2 online). In particular, the
biomass composition of outer and inner cotyledon and radicle/hypocotyl
were estimated based on NMR measurements of organ volumes, as well
as water, lipid, protein, and carbohydrate content (see Supplemental Data
Set 3C online). Photon flux rates were based on the ambient light intensity
Trade-Off between Growth and Storage13 of 16
in the growth chamber (400 µmol photons m22s21), taking into con-
sideration measured light absorbance of pod wall, seed coat, and outer
cotyledons at 32 d after fertilization and measured ETRs. Then, for each
submodel, a fixed value for non-growth-associated ATP drain (ATPdrain)
was determined in same way as described before (Hay and Schwender,
2011a). ATPdrain is a generic reaction summarizing the effect of ATP-
the ATPdrain flux was determined by simulation of dark heterotrophy
(photon uptake flux set to zero) while imposing a carbon balance to the
model as measured before for oilseed rape embryos grown in liquid
cultures in the dark (Goffman et al., 2005). Flux variability was calculated
Flux and Supplemental Data Set 3 online). Flux variability was also cal-
culated as network projection on a lumped reaction network in order to
visualize selected lumped fluxes of central carbon metabolism.
The following materials are available in the online version of this article.
Supplemental Figure 1. Fatty Acid Profile of Various Seed Organs in
B. napus at Mid Storage Stage; Measured as Fatty Acid Methyl Esters
Using Gas Chromatography.
Supplemental Figure 2. Oleosin Deposition in Developing Seeds of B.
Supplemental Figure 3. Spatial and Temporal Pattern of Napin
Accumulation in Developing Seeds of B. napus.
Supplemental Figure 4. Metabolite Distribution in Various Organs of
the B. napus Embryo (30 DAP).
Supplemental Figure 5. Effect of Pod Shading on Lipid Content and
Fatty Acid Composition in Various Components of the B. napus Embryo.
Supplemental Figure 6. Comparison of Biomass Composition and
Steady State Metabolite Levels of B. napus Embryos Grown in Planta
versus in Vitro.
Supplemental Data Set 1. Appendix Proteome.
Supplemental Data Set 2. Appendix Flux.
Supplemental Data Set 3. Flux Values for Three Submodels Repre-
senting Various Embryo Organs.
Supplemental Data Set 4. Flux Values for Outer Cotyledon under
Varying Light Supply from 0 to 2000 µmol Quanta m22s21Ambient
Supplemental Data Set 5. Comparative Analyses of Total Proteins
Extracted from Embryos Grown under Lit and Nonlit Conditions: Image
of 2D IEF/SDS-PAGE, List, and Functional Classification of Proteins.
Supplemental Data Set 6. List of Metabolite Data Measured for
Developing B. napus Embryos.
Supplemental Data Set 7. Comparative Analyses of Total Proteins
Extracted from Embryos Grown in Vitro and in Planta: Image of 2D IEF/
SDS-PAGE, List, and Functional Classification of Proteins.
Supplemental Movie 1. Three-Dimensional Model of Lipid Distribu-
tion in the Embryo of B. napus at Stage III (Mid Storage Phase).
Supplemental Movie 2. Three-Dimensional Model of Lipid Distribu-
tion in the Embryo of B. napus at Stage IV (Late Storage Phase).
Supplemental Movie 3. Three-Dimensional Model of Lipid Distribu-
tion in the Embryo of B. napus at Stage V (Maturation Phase).
Supplemental Movie 4. Example Showing the Reassembling of
Component-Specific Lipid Signal within the Corresponding Volume
(B. napus Seed at Mid Developmental Stage).
Supplemental Movie 5. Three-Dimensional Model of Lipid Distribu-
tion in Cultured Embryos of B. napus.
We thank C. Haase for support in proteomics, Steffen Wagner, Sabine
Herrmann, and Katrin Blaschek for excellent technical assistance, and
Karin Lipfert for artwork. This research was financially supported by
Bayer CropScience. L.B., H.R., and P.M.J. thank the German Federal
Ministry of Education and Research (Deutsche Forschungsgemeinschaft,
BO-1917/4-1) for financial support. J.S. and J.O.H. thank the U.S. De-
partment of Energy (Division of Chemical Sciences, Geosciences, and
Biosciences, Office of Basic Energy Sciences, Field Work Proposal BO-133).
L.B. and H.R. designed the research, performed research, analyzed data,
and wrote the article. T.N. and J.F. performed research (MRI). J.S. and J.
O.H. performed research (FBA). S.S. performed research (proteomics). N.
H. performed research (metabolite analysis). H.T. performed research
(PAM analysis). H.-P.B, P.M.J., P.D., and B.L. analyzed data.
Received March 21, 2013; revised April 27, 2013; accepted May 3, 2013;
published May 24, 2013.
Abbadi, A., and Leckband, G. (2011). Rapeseed breeding for oil
content, quality, and sustainability. Eur. J. Lipid Sci. Technol. 113:
Adamski, N.M., Anastasiou, E., Eriksson, S., O’Neill, C.M., and
Lenhard, M. (2009). Local maternal control of seed size by KLUH/
CYP78A5-dependent growth signaling. Proc. Natl. Acad. Sci. USA
Allen, D.K., Ohlrogge, J.B., and Shachar-Hill, Y. (2009). The role of
light in soybean seed filling metabolism. Plant J. 58: 220–234.
Allen, D.K., and Young, J.D. (2013). Carbon and nitrogen provisions
alter the metabolic flux in developing soybean embryos. Plant
Physiol. 161: 1458–1475.
Alonso, A.P., Dale, V.L., and Shachar-Hill, Y. (2010). Understanding
fatty acid synthesis in developing maize embryos using metabolic
flux analysis. Metab. Eng. 12: 488–497.
Alonso, A.P., Goffman, F.D., Ohlrogge, J.B., and Shachar-Hill, Y.
(2007). Carbon conversion efficiency and central metabolic fluxes in
developing sunflower (Helianthus annuus L.) embryos. Plant J. 52:
Alonso, A.P., Val, D.L., and Shachar-Hill, Y. (2011). Central
metabolic fluxes in the endosperm of developing maize seeds and
their implications for metabolic engineering. Metab. Eng. 13: 96–107.
Andriotis, V.M.E., Pike, M.J., Kular, B., Rawsthorne, S., and Smith,
A.M. (2010). Starch turnover in developing oilseed embryos. New
Phytol. 187: 791–804.
Belmonte, M.F., et al. (2013). Comprehensive developmental profiles
of gene activity in regions and subregions of the Arabidopsis seed.
Proc. Natl. Acad. Sci. USA 110: E435–444.
Baud, S., and Lepiniec, L. (2009). Regulation of de novo fatty acid
synthesis in maturing oilseeds of Arabidopsis. Plant Physiol.
Biochem. 47: 448–455.
14 of 16The Plant Cell
Borisjuk, L., Nguyen, T.H., Neuberger, T., Rutten, T., Tschiersch,
H., Claus, B., Feussner, I., Webb, A.G., Jakob, P., Weber, H.,
Wobus, U., and Rolletschek, H. (2005). Gradients of lipid storage,
photosynthesis and plastid differentiation in developing soybean
seeds. New Phytol. 167: 761–776.
Borisjuk, L., Rolletschek, H., Fuchs, J., Melkus, G., and Neuberger,
T. (2011). Recent applications of ‘low field’ and ‘high field’ magnetic
resonance in seed research. Materials 4: 1426–1439.
Borisjuk, L., Rolletschek, H., and Neuberger, T. (2012). Surveying
the plant’s world by magnetic resonance imaging. Plant J. 70: 129–146.
Chen, M., Mooney, B.P., Hajduch, M., Joshi, T., Zhou, M., Xu, D.,
and Thelen, J.J. (2009). System analysis of an Arabidopsis mutant
altered in de novo fatty acid synthesis reveals diverse changes in
seed composition and metabolism. Plant Physiol. 150: 27–41.
Da Silva, P.M.F.R., Eastmond, P.J., Hill, L.M., Smith, A.M., and
Rawsthorne, S. (1997). Starch metabolism in developing embryos
of oilseed rape. Planta 203: 480–487.
De Smet, I., Lau, S., Mayer, U., and Jürgens, G. (2010). Embryogenesis -
The humble beginnings of plant life. Plant J. 61: 959–970.
Fernie, A.R., and Stitt, M. (2012). On the discordance of metabolomics
with proteomics and transcriptomics: Coping with increasing complexity
in logic, chemistry, and network interactions scientific correspondence.
Plant Physiol. 158: 1139–1145.
Fuchs, J., Neuberger, T., Rolletschek, H., Schiebold, S., Nguyen,
T.H., Borisjuk, N., Börner, A., Melkus, G., Jakob, P., and Borisjuk,
L. (2013). A noninvasive platform for imaging and quantifying oil
storage in submillimeter tobacco seed. Plant Physiol. 161: 583–593.
Garcia, D., Saingery, V., Chambrier, P., Mayer, U., Jürgens, G., and
Berger, F. (2003). Arabidopsis haiku mutants reveal new controls of
seed size by endosperm. Plant Physiol. 131: 1661–1670.
Gegas, V.C., Nazari, A., Griffiths, S., Simmonds, J., Fish, L., Orford,
S., Sayers, L., Doonan, J.H., and Snape, J.W. (2010). A genetic
framework for grain size and shape variation in wheat. Plant Cell 22:
Goffman, F.D., Alonso, A.P., Schwender, J., Shachar-Hill, Y., and
Ohlrogge, J.B. (2005). Light enables a very high efficiency of
carbon storage in developing embryos of rapeseed. Plant Physiol.
Grami, B., Baker, R.J., and Stefansson, B.R. (1977). Genetics of
protein and oil content in summer rape: heritability, number of
effective factors, and correlations. Can. J. Plant Sci. 57: 937–943.
Hay, J.O., and Schwender, J. (2011a). Metabolic network recon-
struction and flux variability analysis of storage synthesis in
developing oilseed rape (Brassica napus L.) embryos. Plant J. 67: 526–
Hay, J.O., and Schwender, J. (2011b). Computational analysis of
storage synthesis in developing Brassica napus L. (oilseed rape)
embryos: Flux variability analysis in relation to ¹³C metabolic flux
analysis. Plant J. 67: 513–525.
Hayden, D.M., Rolletschek, H., Borisjuk, L., Corwin, J., Kliebenstein,
D.J., Grimberg, A., Stymne, S., and Dehesh, K. (2011). Cofactome
analyses reveal enhanced flux of carbon into oil for potential biofuel
production. Plant J. 67: 1018–1028.
Horn, P.J., Korte, A.R., Neogi, P.B., Love, E., Fuchs, J., Strupat, K.,
Borisjuk, L., Shulaev, V., Lee, Y.-J., and Chapman, K.D. (2012).
Spatial mapping of lipids at cellular resolution in embryos of cotton.
Plant Cell 24: 622–636.
Ingouff, M., Jullien, P.E., and Berger, F. (2006). The female
gametophyte and the endosperm control cell proliferation and
differentiation of the seed coat in Arabidopsis. Plant Cell 18: 3491–
Junker, B.H., Lonien, J., Heady, L.E., Rogers, A., and Schwender,
J. (2007). Parallel determination of enzyme activities and in vivo
fluxes in Brassica napus embryos grown on organic or inorganic
nitrogen source. Phytochemistry 68: 2232–2242.
Lee, C.P., Eubel, H., and Millar, A.H. (2010). Diurnal changes in
mitochondrial function reveal daily optimization of light and dark
respiratory metabolism in Arabidopsis. Mol. Cell. Proteomics 9:
Li, Y., Beisson, F., Pollard, M., and Ohlrogge, J.B. (2006). Oil
content of Arabidopsis seeds: The influence of seed anatomy, light
and plant-to-plant variation. Phytochemistry 67: 904–915.
Lonien, J., and Schwender, J. (2009). Analysis of metabolic flux
phenotypes for two Arabidopsis mutants with severe impairment in
seed storage lipid synthesis. Plant Physiol. 151: 1617–1634.
Melkus, G., et al. (2011). Dynamic ¹³C/¹ H NMR imaging uncovers
sugar allocation in the living seed. Plant Biotechnol. J. 9: 1022–1037.
Melkus, G., Rolletschek, H., Radchuk, R., Fuchs, J., Rutten, T.,
Wobus, U., Altmann, T., Jakob, P.M., and Borisjuk, L. (2009). The
metabolic role of the legume endosperm: A noninvasive imaging
study. Plant Physiol. 151: 1139–1154.
Meyer, L.J., Gao, J., Xu, D., and Thelen, J.J. (2012). Phosphoproteomic
analysis of seed maturation in Arabidopsis, rapeseed, and soybean.
Plant Physiol. 159: 517–528.
Moles, A.T., Ackerly, D.D., Webb, C.O., Tweddle, J.C., Dickie, J.B.,
and Westoby, M. (2005). A brief history of seed size. Science 307:
Muller-Landau, H.C. (2010). The tolerance-fecundity trade-off and
the maintenance of diversity in seed size. Proc. Natl. Acad. Sci. USA
Murphy, D.J., and Cummins, I. (1989). Biosynthesis of seed storage
products during embryogenesis in rapeseed, Brassica napus. J.
Plant Physiol. 135: 63–69.
Neuberger, T., Rolletschek, H., Webb, A., and Borisjuk, L. (2009).
Non-invasive mapping of lipids in plant tissue using magnetic res-
onance imaging. In Lipidomics, Volume 1: Methods and Protocols,
D. Armstrong, ed (New York: Humana Press) pp. 485–496.
Niu, Y., Wu, G.Z., Ye, R., Lin, W.-H., Shi, Q.-M., Xue, L.-J., Xu, X.-D.,
Li, Y., Du, Y.-G., and Xue, H.-W. (2009). Global analysis of gene
expression profiles in Brassica napus developing seeds reveals
a conserved lipid metabolism regulation with Arabidopsis thaliana.
Mol. Plant 2: 1107–1122.
North, H., et al. (2010). Arabidopsis seed secrets unraveled after
a decade of genetic and omics-driven research. Plant J. 61: 971–981.
Nunes-Nesi, A., Araújo, W.L., Obata, T., and Fernie, A.R. (February
22, 2013). Regulation of the mitochondrial tricarboxylic acid cycle.
Curr. Opin. Plant Biol. .
O’Grady, J., Schwender, J., Shachar-Hill, Y., and Morgan, J.A.
(2012). Metabolic cartography: Experimental quantification of
metabolic fluxes from isotopic labelling studies. J. Exp. Bot. 63:
Ohlrogge, J., Allen, D., Berguson, B., Dellapenna, D., Shachar-Hill,
Y., and Stymne, S. (2009). Energy. Driving on biomass. Science
Ohto, M.A., Fischer, R.L., Goldberg, R.B., Nakamura, K., and
Harada, J.J. (2005). Control of seed mass by APETALA2. Proc.
Natl. Acad. Sci. USA 102: 3123–3128.
Oliveira, A.P., Ludwig, C., Picotti, P., Kogadeeva, M., Aebersold,
R., and Sauer, U. (2012). Regulation of yeast central metabolism by
enzyme phosphorylation. Mol. Syst. Biol. 8: 623.
Ponnu, J., Wahl, V., and Schmid, M. (2011). Trehalose-6-phosphate:
Connecting plant metabolism and development. Front. Plant Sci. 2: 70.
Radchuk, V., Kumlehn, J., Rutten, T., Sreenivasulu, N., Radchuk,
R., Rolletschek, H., Herrfurth, C., Feussner, I., and Borisjuk, L.
(2012). Fertility in barley flowers depends on Jekyll functions in male
and female sporophytes. New Phytol. 194: 142–157.
Trade-Off between Growth and Storage15 of 16
Ramli, U.S., Salas, J.J., Quant, P.A., and Harwood, J.L. (2009). Use
of metabolic control analysis to give quantitative information on
control of lipid biosynthesis in the important oil crop, Elaeis
guineensis (oilpalm). New Phytol. 184: 330–339.
Rolletschek, H., Koch, K., Wobus, U., and Borisjuk, L. (2005).
Positional cues for the starch/lipid balance in maize kernels and
resource partitioning to the embryo. Plant J. 42: 69–83.
Rolletschek, H., Melkus, G., Grafahrend-Belau, E., Fuchs, J.,
Heinzel, N., Schreiber, F., Jakob, P.M., and Borisjuk, L. (2011).
Combined noninvasive imaging and modeling approaches reveal
metabolic compartmentation in the barley endosperm. Plant Cell
Ruuska, S.A., Schwender, J., and Ohlrogge, J.B. (2004). The
capacity of green oilseeds to utilize photosynthesis to drive
biosynthetic processes. Plant Physiol. 136: 2700–2709.
Sanjaya, D., Durrett, T.P., Weise, S.E., and Benning, C. (2011).
Increasing the energy density of vegetative tissues by diverting
carbon from starch to oil biosynthesis in transgenic Arabidopsis.
Plant Biotechnol. J. 9: 874–883.
Schiebold, S., Tschiersch, H., Borisjuk, L., Heinzel, N., Radchuk,
R., and Rolletschek, H. (2011). A novel procedure for the
transcripts of laser microdissected seed tissues of Brassica napus.
Plant Methods 7: 19.
Schwender, J. (2008). Metabolic flux analysis as a tool in metabolic
engineering of plants. Curr. Opin. Biotechnol. 19: 131–137.
Schwender, J., Goffman, F., Ohlrogge, J.B., and Shachar-Hill, Y.
(2004). Rubisco without the Calvin cycle improves the carbon
efficiency of developing green seeds. Nature 432: 779–782.
Schwender, J., Shachar-Hill, Y., and Ohlrogge, J.B. (2006).
Mitochondrial metabolism in developing embryos of Brassica
napus. J. Biol. Chem. 281: 34040–34047.
Shen, B., Allen, W.B., Zheng, P., Li, C., Glassman, K., Ranch, J.,
Nubel, D., and Tarczynski, M.C. (2010). Expression of ZmLEC1
and ZmWRI1 increases seed oil production in maize. Plant Physiol.
Transcriptional profiling of the Arabidopsis embryo. Plant Physiol.
Sun, X., Shantharaj, D., Kang, X., and Ni, M. (2010). Transcriptional
and hormonal signaling control of Arabidopsis seed development.
Curr. Opin. Plant Biol. 13: 611–620.
storage products and
Sweetlove, L.J., and Ratcliffe, R.G. (2011). Flux-balance modeling of
plant metabolism. Front. Plant Sci. 38: 1–10.
Tang, M., Guschina, I.A., O’Hara, P., Slabas, A.R., Quant, P.A.,
Fawcett, T., and Harwood, J.L. (2012). Metabolic control analysis
of developing oilseed rape (Brassica napus cv Westar) embryos shows
that lipid assembly exerts significant control over oil accumulation. New
Phytol. 196: 414–426.
Taylor, D.C., et al. (2009). Molecular modification of triacylglycerol
accumulation by over-expression of DGAT1 to produce canola with
increased seed oil content under field conditions. Botany 87: 533–543.
Tiedemann, J., Rutten, T., Mönke, G., Vorwieger, A., Rolletschek, H.,
Meissner, D., Milkowski, C., Petereck, S., Mock, H.P., Zank, T., and
Bäumlein, H. (2008). Dissection of a complex seed phenotype: Novel
insights of FUSCA3 regulated developmental processes. Dev. Biol. 317:
Tschiersch, H., Borisjuk, L., Rutten, T., and Rolletschek, H. (2011).
Gradients of seed photosynthesis and its role for oxygen balancing.
Biosystems 103: 302–308.
Troncoso-Ponce, M.A., Kilaru, A., Cao, X., Durrett, T.P., Fan, J.,
Jensen, J.K., Thrower, N.A., Pauly, M., Wilkerson, C., and
Ohlrogge, J.B. (2011). Comparative deep transcriptional profiling of
four developing oilseeds. Plant J. 68: 1014–1027.
Verboven, P., Herremans, E., Borisjuk, L., Helfen, L., Ho, Q.T.,
Tschiersch, H., Fuchs, J., Nicolaï, B.M., and Rolletschek, H. (May
21, 2013). Void space inside the developing seed of Brassica napus
and the modelling of its function. New Phytologist (doi: 10.1111/
Vigeolas, H., Möhlmann, T., Martini, N., Neuhaus, H.E., and
Geigenberger, P. (2004). Embryo-specific reduction of ADP-Glc
pyrophosphorylase leads to an inhibition of starch synthesis and
a delay in oil accumulation in developing seeds of oilseed rape.
Plant Physiol. 136: 2676–2686.
Wallis, J.G., and Browse, J. (2010). Lipid biochemists salute the
genome. Plant J. 61: 1092–1106.
Weselake, R.J., Taylor, D.C., Rahman, M.H., Shah, S., Laroche, A.,
McVetty, P.B.E., and Harwood, J.L. (2009). Increasing the flow of
carbon into seed oil. Biotechnol. Adv. 27: 866–878.
Xiang, D., Venglat, P., Tibiche, C., Yang, H., Risseeuw, E., Cao, Y.,
Babic, V., Cloutier, M., Keller, W., Wang, E., Selvaraj, G., and
Datla, R. (2011). Genome-wide analysis reveals gene expression
and metabolic network dynamics during embryo development in
Arabidopsis. Plant Physiol. 156: 346–356.
16 of 16The Plant Cell
Page 17 Download full-text