Combined Noninvasive Imaging and Modeling ApproachesReveal Metabolic Compartmentation in theBarley Endosperm W OA

Hardy Rolletschek,a,1 Gerd Melkus,b,1 Eva Grafahrend-Belau,a Johannes Fuchs,a,c Nicolas Heinzel,a

Falk Schreiber,a Peter M. Jakob,c and Ljudmilla Borisjuka,2

a Leibniz-Institut fur Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germanyb University of California–San Francisco, Radiology and Biomedical Imaging, San Francisco, California 94107c University of Wurzburg, Institute of Experimental Physics 5, 97074 Wuerzburg, Germany

The starchy endosperm of cereals is a priori taken as a metabolically uniform tissue. By applying a noninvasive assay based

on 13C/1H-magnetic resonance imaging (MRI) to barley (Hordeum vulgare) grains, we uncovered metabolic compartmen-

tation in the endosperm. 13C-Suc feeding during grain filling showed that the primary site of Ala synthesis was the central

region of the endosperm, the part of the caryopsis experiencing the highest level of hypoxia. Region-specific metabolism in

the endosperm was characterized by flux balance analysis (FBA) and metabolite profiling. FBA predicts that in the central

region of the endosperm, the tricarboxylic acid cycle shifts to a noncyclic mode, accompanied by elevated glycolytic flux

and the accumulation of Ala. The metabolic compartmentation within the endosperm is advantageous for the grain’s carbon

and energy economy, with a prominent role being played by Ala aminotransferase. An investigation of caryopses with a

genetically perturbed tissue pattern demonstrated that Ala accumulation is a consequence of oxygen status, rather than

being either tissue specific or dependent on the supply of Suc. Hence the 13C-Ala gradient can be used as an in vivo marker

for hypoxia. The combination of MRI and metabolic modeling offers opportunities for the noninvasive analysis of metabolic

compartmentation in plants.

INTRODUCTION

Cereal grains form the basis of most of the human diet and most

animal feed. Among grains, barley (Hordeum vulgare) is widely

cultivated in all temperate regions and ranks fourth in worldwide

production. Because of its importance in agriculture, the barley

genome is currently being sequenced (Schulte et al., 2009; http://

barleygenome.org), and the development of the barley caryop-

sis, or grain, has been studied in some detail (Sreenivasulu et al.,

2010). Apart from the transfer cells and aleurone layer, the barley

endosperm appears quite homogeneous. However, in vivo

tracking during grain filling has established regional variation in

the supply of Suc to the endosperm (Melkus et al., 2011), clear

developmental gradients (Sabelli and Larkins, 2009), and uneven

levels of hypoxia within the caryopsis (Rolletschek et al., 2004).

These localized differences in the internal environment of the

endosperm suggest a compartmentation of metabolic activity,

although this is difficult to detect directly without access to

markers for distinct biochemical events.

Alanine aminotransferase (Ala-AT; EC 2.6.1.2) catalyzes the

interconversion of pyruvate to Ala and is regarded as a critical

branch point separating aerobic from anaerobic metabolism

(Good and Crosby, 1989; Miyashita et al., 2007; Miyashita and

Good, 2008; Rocha et al., 2010). The heterologous expression of

barley Ala-AT produces a marked improvement in the nitrogen

use efficiency of canola (Brassica napus; Good et al., 2007) and

rice (Oryza sativa; Beatty et al., 2009). It was argued that this has

revolutionary importance for future crop production (Ridley,

2009). In the barley caryopsis, the gene encoding Ala-AT be-

comes more highly expressed as the assimilate storage phase

proceeds (Sreenivasulu et al., 2006), corresponding well with

the rising level of internal hypoxia (Rolletschek et al., 2004). Its

expression is higher in the endosperm than in the pericarp

(Sreenivasulu et al., 2006), both in barley and in rice grains

(Kikuchi et al., 1999). The in vivo function of this enzyme appears

to be to catalyze the synthesis of Ala, but the reversibility of this

reaction complicates the picture because its direction is depen-

dent on the local oxygen status and the relative concentrations of

Ala and pyruvate present. Ala-AT activity has long been used in

the medical field as a marker for certain diseases (Salaspuro,

1989;Nyblomet al., 2004) and inflammatory responses (Gelderblom

et al., 2008) and can be detected by noninvasive methods.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy

allows for the noninvasive in vivo detection, quantification, and

mapping of certain metabolites in plants (Ratcliffe et al., 2001;

Kockenberger et al., 2004). For example, major assimilates, such

as Suc and Gln, can be detected noninvasively in seeds of pea

1 These authors contributed equally to this work.2 Address correspondence to borysyuk@ipk-gatersleben.de.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Ljudmilla Borisjuk(borysyuk@ipk-gatersleben.de).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.111.087015

The Plant Cell, Vol. 23: 3041–3054, August 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.

(Pisum sativum) using localized 1H-NMR spectroscopy and

chemical shift imaging (Melkus et al., 2009). The advantage of

using the signal from 1H instead of other nuclei, such as 13C or15N, is its higher sensitivity, but its utility is limited to measuring

steady state levels. By contrast, 13C NMR can be combined with

the feeding of 13C labeled substrates to track their localization

along with their metabolic products. By combining NMR spec-

troscopy and imaging, it is possible to acquire metabolic and

spatial information of the 13C enrichedmolecule and its products

in the same experiment. To further improve the sensitivity of

detection of the 13C nucleus, different inverse detection schemes

were developed for preclinical animal and human applications

(Bax et al., 1983, Rothman et al., 1985, 1992). These pulse

sequences provide different strategies to edit and detect the

protons connected to the 13C nuclei and reach therefore in

general higher sensitivity than direct 13C detection (Novotny

et al., 1990; Heidenreich et al., 1998; de Graaf et al., 2003).

Here, we applied the gradient enhanced heteronuclear multi-

ple quantum coherence (geHMQC; Hurd and John, 1991) se-

quence to enable the detection of 13C-Ala, derived from a supply

of 13C-Suc to a barley plant. Noninvasivemetabolite imagingwas

able to demonstrate that 13C-Ala synthesis is restricted to the

innermost, most hypoxic region of the endosperm. Biochemical

and flux balance analysis (FBA) have also been applied to

interpret this spatial arrangement, producing a spatially resolved

metabolic model of the starchy endosperm in barley.

RESULTS

Experimental Design for Localizing Ala Synthesis in

the Caryopsis

To localize Ala synthesis, we first needed to determine the best

precursor form and route to deliver the 13C label. Analysis of

caryopsis sugar composition indicates a characteristic develop-

mental shift from mainly hexoses to mainly Suc (see Supple-

mental Figure 1 online). Ala was the most prominent free amino

acid present, at ;30% of the total pool. The Ala present in the

caryopsis included a portion delivered through the vascular

strands in the crease vein and a portion synthesized within the

caryopsis. The latter was detected by means of feeding 13C-Suc

to the intact caryopsis via the stem (Figure 1A; for details, see

Methods). The extent and kinetics of the uptake of the labeled

Suc and its subsequent synthesis to Ala was determined by

applying mass spectrometry. The proportion of 13C-Suc present

rose significantly within 12 h of feeding (Figure 1B), and the

accumulation of labeled Ala followed a similar time course. The

presence of labeled Glc and Fru remained low.

For noninvasive studies, we applied 13C/1H-magnetic reso-

nance imaging (MRI) to visualize the anatomy of the ear (see

Supplemental Movie 1 online) and the allocation of 13C-Suc (see

Supplemental Movie 2 online). Dynamic MRI demonstrates that

Suc passes via crease vein and nucellar projection toward the

endosperm and spreads centrifugally within endosperm (for

more details, see Melkus et al., 2011).

In addition, we optimized the detection method to allow us

to distinguish 13C-Ala from 13C-Suc and other molecules. The

noninvasive localization of de novo Ala synthesis was achieved

using the geHMQC editing scheme combinedwith chemical shift

imaging for spatial localization (see Supplemental Figure 2A

online). Sufficient water suppression was achieved with a fre-

quency selective water presaturation module (VAPOR suppres-

sion; Tkac et al., 1999). To improve the signal-to-noise ratio

(SNR), the 13C nuclei were irradiated with a Malcolm Levitt

decoupling pulse scheme, while the 1H signal was acquired (see

Methods). Supplemental Figures 2B and 2C show spectra ac-

quired with the geHMQC from 100 mM solutions of 13C-Suc and13C-Ala. The resonances of the protons from the 13C-Suc are

visible in the spectral region between 3.4 and 4.2 ppm, and the

anomeric hydrogen on the pyranose ring is detectable at 5.4 ppm

(see Supplemental Figure 2B online). The protons connected to13C-Ala resonate at 1.48 ppm (methyl group) and 3.78 ppm

(methin group) (see Supplemental Figure 2C online). The methyl

resonance from Ala is therefore well separated from the Suc

signals in the spectrum, allowing the separation of the two

Figure 1. Feeding of 13C-Suc to the Endosperm.

(A) Schematic representation of 13C-Suc feeding to the barley ear; the

cage indicates the placement of the NMR coil used for metabolite

imaging.

(B) The ratio of 13C-to-12C metabolites in grain extracts during feeding,

as measured by LC-MS.

(C) A slice of a three-dimensional reconstruction of an ear showing the

pericarp (p), endosperm (en), and nucellar projection (np) of an intact

caryopsis, as measured by NMR (see Supplemental Movie 1 online).

(D) The scheme indicates the direction of Suc flow within the endosperm

(see Supplemental Movie 2 online). The three-dimensional model of

caryopsis is based on micro-computer tomography, and flow direction

is based on NMR.

3042 The Plant Cell

metabolites also in the in vivo spectra. Figure 2A shows a 1H

reference image of the barley caryopsis, where the locations of

two voxels from the geHMQC sequence are indicated. Typical

geHMQC spectra are shown in Figures 2D and 2E. The spectrum

from the vascular region (Figure 2D) shows intense signal from

the delivered 13C-Suc, while signals from other 13C metabolites

are not visible. The central region of the endosperm features a

major 13C-Ala resonance (Figure 2E). This signal is well sepa-

rated from resonances of 13C-Suc. We conclude that the 13C-Ala

was derived from the incoming 13C-Suc.

The Primary Site of Ala Synthesis Is the Central Endosperm

To determine where Ala is synthesized in the endosperm, we

used seeds fed with 13C-Suc and determined the localization of

labeled Suc and Ala. The metabolic Suc image was calculated

from the Suc resonances, avoiding the spectral region around

3.78 ppm to prevent signal contamination arising from the Ala

methin group (see Supplemental Figure 2B online). The distribu-

tion of Ala was computed from the Ala methyl resonance at 1.48

ppm (see Supplemental Figure 2C online). Two sites of high Suc

levels were detected, one at the crease vein/nucellar projection

and a second, larger onewithin the central part of the endosperm

(Figure 2B). Ala also accumulated in this latter region, but not in

the pericarp or elsewhere in the peripheral region of the endo-

sperm (Figure 2C). Over the course of the feeding experiment,

the Ala signal gained in intensity within the central part of the

endosperm, forming steep gradients toward the periphery. Thus,

the major site of Ala synthesis clearly lay within the central part of

the endosperm, a conclusion borne out by a comparison of Ala

accumulation between the inner and outer regions of the endo-

sperm (see below).

Ala Gradients Reflect the Local Oxygen State of

the Endosperm

The oxygen level within the developing caryopsis has been

examined in detail by Rolletschek et al. (2004). They showed that

the central region of the endosperm is highly hypoxic (falling to <5

mM oxygen), while in the photosynthetically active portion of the

pericarp, the oxygen concentration is, if anything, oversaturated

under lit conditions (Figure 3B). In the absence of light, the

conditions in the central region of the endosperm tend toward

stronger hypoxia (<2 mM oxygen), unlike in the periphery, where

hypoxia does not occur (Figure 3C). Ala synthesis is therefore

largely confined to the most hypoxic region of the endosperm.

The gradient in Ala also colocalized with pattern of cell ex-

pansion (see Supplemental Figure 3A online). The cell size is

maximal within the central hypoxic region and decreases toward

the periphery. We did not detect any structural barriers between

these regions, but rather multiple plasmodesmata connecting

cells within the entire starchy endosperm (see Supplemental

Figures 3B and 3C online).

Steady State Metabolite Levels Differ in Inner versus Outer

Regions of Starchy Endosperm

To determine whether other metabolites showed a similar gra-

dient to Ala, we next compared the inner and outer endosperm

by liquid chromatography–mass spectrometry (LC-MS). The

Figure 2. 13C Metabolic Imaging Using geHMQC.

(A) 1H-reference image of the barley caryopsis. The two voxels D and E indicate the location of the geHMQC spectra shown in (D) and (E).

(B) Allocation of 13C-Suc after 12 h of feeding with 100 mM 13C-Suc.

(C) Ala distribution after 12 h of feeding with 100 mM 13C-Suc.

(D) geHMQC spectrum from the pericarp region (voxel D in [A]).

(E) geHMQC spectrum from the endosperm (voxel E in [A]).

Compartmentation of Cereal Endosperm 3043

Figure 3. Oxygen Distribution Maps and Metabolite Patterns in the Barley Endosperm.

3044 The Plant Cell

NMR-based metabolite imaging indicated some spatial hetero-

geneity within the endosperm for sugar to amino acid conver-

sion, and this conclusion was supported by the LC-MS–based

comparison of steady state metabolite levels between the en-

dosperm’s inner and outer regions (Figure 3). These data dem-

onstrated that the intermediate products of both Suc breakdown

(i.e., hexoses and hexose phosphates) and glycolysis persisted

at higher levels in the peripheral region of the endosperm.

Likewise, several tricarboxylic acid cycle–related organic acids,

with the exception of fumarate, were distributed in this manner.

Most of the free amino acids were evenly distributed throughout

the endosperm, with the exception of Ala and Gly, accumulating

in the inner regions, and homocysteine (and also 5-methyl-

tetrahydrofolate), which concentrated in the peripheral region.

Despite remarkable differences in oxygen influx (corresponding

to the respiratory oxygen consumption), those intermediates

related to energy metabolism (except NADP) maintained com-

parable steady state levels across the endosperm. The picture is

therefore onewheremetabolite levels and presumablymetabolic

pathway activity differs substantially across the starchy endo-

sperm.

The Response of Ala Metabolism to Variation in the Supply

of Oxygen

We next tested whether Ala synthesis changed with different

oxygen levels, contrasting intact ears subjected to either normal

(21 kPa) or elevated (42 kPa) levels of oxygen. The level of Ala

declined as the oxygen supply was increased (Figure 4, top

panel), as did the levels of phosphoenolpyruvate, pyruvate, Glu,

and Asp. By contrast, those of oxoglutarate and oxaloacetate

(acceptor molecules for the amino group in the transamination

reactions catalyzed by Ala-AT and aspartate aminotransferase

[Asp-AT]) remained unchanged and ammonia increased (see

Supplemental Table 1 online). There was no perceptible shift in

the fermentation products lactate and ethanol. Ala-AT activity, as

estimated by its peak catalytic activity (Vmax) in tissue extracts,

was also unaffected by the increased supply of oxygen (Figure 4),

nor likewise were Asp-AT (related to Ala metabolism) and the

fermentation enzymes pyruvate decarboxylase, alcohol dehy-

drogenase, and lactate dehydrogenase. The oxygen-dependent

shifts in metabolite pools indicate altered activities in the path-

ways related to Ala metabolism. None of the genes encoding

enzymes above was transcriptionally induced, as assayed by

macroarray analysis (V. Radchuk, personal communication). It

appeared that the regulation of Ala metabolism is rather inde-

pendent of transcriptional/translational control.

To elucidate the in vivo enzymatic activities, we then attempted

a modeling approach (FBA) based on the barley caryopsis met-

abolic network established by Grafahrend-Belau et al. (2009a).

Ala-AT function was simulated using the synthesis/degradation

of Ala in response to changes in the oxygen supply (Figure 4,

bottom panel). This predicted that Ala accumulates under hy-

poxic conditions, producing an Ala gradient opposite to that of

oxygen. As the supply of oxygen was increased, the accumula-

tion of Ala falls as a result of a reduced flux of both the cytosolic

and mitochondrial isoforms of Ala-AT, both of which direct Ala

synthesis. At an oxygen level above 8 mmol g dry weight (DW)21

h21, Ala was no longer accumulated due to the deactivation of

mAla-AT and the reverse action of cAla-AT, which converts Ala

back to pyruvate.

Simulation of Compartment-Specific Metabolism in

the Caryopsis

Based on the above FBA, a simulation was then performed to

model metabolic flux distributions appearing in central versus

peripheral endosperm (Figure 5; see Supplemental Data Set 2

online). What follows is a description of the tissue-specific flux

maps, with the major changes to metabolic behavior being

highlighted.

Metabolism in the Central Hypoxic Endosperm

The metabolic flux distribution in the central endosperm reflects

the outcome of limited respiration, the activation of fermentation,

and the stimulation of glycolysis (fermentative Pasteur effect).

The TCA cycle shifts to a noncyclic mode due to inactive

succinate dehydrogenase (SDH). The activation of cAla-ATA

and mAla-ATA results in the accumulation of Ala, the use of

pyruvate, and the production of oxoglutarate, which can act as a

cosubstrate for glutamate dehydrogenase (GDH) in the mito-

chondria. As themitochondrial electron transport chain is limited,

GDH represents the main source of mitochondrial NAD+ regen-

eration, providing the redox equivalents required for the TCA

cycle. In the cytosol, oxoglutarate reacts with aspartate to form

Glu and oxaloacetate through the action of cAsp-AT. Cytosolic

malate dehydrogenase (MDH), which catalyzes the reaction from

oxaloacetate to malate, is the main source of cytosolic NAD+

regeneration. Cytosolic NAD+ needs to be constantly regener-

ated to permit glycolysis, the primary source of energy under

hypoxia. In the cytosol, the cytosolic MDH–mediated regenera-

tion of NAD+ is boosted by the import of oxaloacetate synthe-

sized in the mitochondria by mitochondrial MDH. In addition to

Figure 3. (continued).

(A) Distribution of chlorophyll (in red) measured in a cross section of caryopsis using laser scanning microscopy.

(B) and (C) Spatial distribution of oxygen in the endosperm under lit and nonlit conditions, respectively; the color bar indicates the oxygen concentration

in mM. ch, chlorenchyma; en, endosperm; ev, endospermal vacuole; p, pericarp.

(D) Steady state metabolite levels in the central versus the peripheral regions of the endosperm, as measured by LC-MS. Data are in the form of mean6

SE (n = 6). Stars indicate statistically significant (P < 0.05) differences according to a t test. Metabolite values are listed in Supplemental Data Set 1 online.

DHAP, dihydroxyacetone-P; PGA, phosphoglycerate; PEP, phosphoenolpyruvate; SSA, succinic semialdehyde; GabaT, g-aminobutyric acid; THF,

tetrahydrofolate.

Compartmentation of Cereal Endosperm 3045

Ala accumulation, the reverse action of fumarase acting on

malate results in the accumulation of fumarate. There is a minor

accumulation of succinate via TCA cycle reactions, but the Gaba

shunt is not involved. Neither the Gaba shunt nor GAD are active,

and there is no accumulation of Gaba.

Metabolism in the Peripheral Aerobic Endosperm

Metabolism in the peripheral endosperm is characterized mainly

by elevated TCA cycle activity, with little or no fermentation, and

decline in glycolytic flux. Thereby, the TCA pathway follows the

common cyclic mode. Neither Ala, fumarate, nor succinate

accumulates in this part of the endosperm. Ala serves either as

a precursor for protein storage synthesis or is converted to

pyruvate by cAla-AT. mAla-AT is no longer active. The induction

of the anaplerotic reaction of phosphoenolpyruvate carboxylase

(PEPC) results in the allocation of carbon skeletons required for

storage product synthesis. In the cytosol, the flux via MDH is

strongly reduced, reflecting a much reduced requirement for

NAD+ regeneration, while in themitochondria, themain source of

NAD+ regeneration is represented by oxidative phosphorylation,

with GDH acting in the direction of NADH synthesis.

Figure 4. Regulation of Ala Metabolism in the Endosperm in Response to a Changing Oxygen Supply.

(A) Steady state metabolite levels and enzyme activites (Vmax) in endosperms developing under ambient and elevated levels of oxygen availability. Stars

indicate statistically significant (P < 0.05) differences according to a t test (n = 6). ADH, alcohol dehydrogenase; GabaT, g-aminobutyric acid

aminotransferase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; PK, pyruvate kinase; SSA, succinic semialdehyde; n.d., not determined.

Ethanol and SSA were undetectable in seed extracts. Metabolite and enzyme data are also given in Supplemental Table 1 online.

(B) Simulation of Ala metabolism under varying oxygen supply based on FBA. DW, dry weight.

3046 The Plant Cell

The FBA also suggests other compartment-specific fea-

tures. For example, while there is no PEPC activity in the central

endosperm, this enzyme is active in the peripheral endo-

sperm (Figure 5). Localization of PEPC in peripheral cary-

opsis tissues (Macnicol and Jacobsen, 1992; Gonzalez et al.,

1998) is in aggreement with this prediction. Our data can also

be taken to suggest that the Vmax values of the various

enzymes (see Supplemental Table 1 online) are much higher

than is necessary to satisfy the endosperm’s in vivo require-

ments (see Supplemental Data Set 2 online), as has also

been observed in embryos of oilseed rape (Junker et al.,

2007).

Suc and Ala Distribution in Plants Engineered to Have

Altered Suc Allocation

Wenext examinedwhether the Ala distribution is altered in plants

with altered Suc distribution. The gene Jekyll controls cell differ-

entiation and cell death within the nucellar projection of caryop-

sis (Radchuk et al., 2006) and in so doing also affects Suc release

from the pericarp into the endosperm. When Jekyll was down-

regulated by means of RNA interference, the release rate of Suc

was greatly reduced (Melkus et al., 2011). A strong repression of

Jekyll (by ;90%) resulted in a switch from cell death to callus-

like growth in the nucellar projection, which expanded and

Figure 5. Flux Maps Depicting the Key Fluxes within Primary Metabolism of the Endosperm.

(A) A three-dimensional image of the caryopsis depicting central and peripheral regions of the endosperm.

(B) and (C) A flux distribution map of the central (hypoxic region; [B]) and the peripheral (aerobic region; [C]) endosperm. The thickness of each arrow

corresponds to the flux value (data also supplied in Supplemental Data Set 2 online). Orange indicates an accumulation of the respective metabolite

under the prevailing conditions.

Compartmentation of Cereal Endosperm 3047

eventually replaced the starchy endosperm (Figure 6A). As it is

enclosed by a pericarp/cuticle, this callus-like tissue readily

becomes hypoxic, with the level of oxygen in the central part of

these caryopses (as measured by a microsensor) falling to 1.560.6 mM. The application of 13C/1H-MRI analysis on these cary-

opses showed that the distribution of Suc was altered, while that

of Ala was left unchanged (Figure 6B). This observation was

taken to show that Ala synthesis is related to the hypoxic status

of the tissue, rather than being dependent on either tissue type or

the pattern of Suc supply.

DISCUSSION

13C/1H-NMR and the in Vivo Imaging of Plant Metabolism

The use of NMR to visualize and quantify metabolites in planta

remains challenging. The present approach has relied on a

spectral editing scheme: the geHMQC sequence used the 1JCH-

coupling to detect the protons attached to 13C nuclei, while at the

same time other magnetization was dephased by pulsed mag-

netic field gradients (Hurd and John, 1991; Tse et al., 1996). This

method allowed the monitoring of 13C-Ala accumulation with a

high level of sensitivity, which in turn demonstrated the localized

synthesis of Ala fromSuc fed to the plant. The approach provides

several major advantages over available alternatives. First, the

assay is noninvasive, avoiding the artifacts arising from sample

preparation; second, apart from complications associated with

feeding the plant with 13C-labeled Suc, the outcomes are faithful

to the in vivo situation, in terms of the presence of endogenous

gradients in the concentrations of metabolites and oxygen, and

the compartmentation structure of the caryopsis remains undis-

turbed; third, the spatial resolution of the 13C-Ala mapping is

sufficient to allow the detection of tissue-specific differences in

metabolism; fourth, the assay times (;20 min) offer a means to

observe the kinetics of 13C-Ala uptake, synthesis, and degrada-

tion; fifth, the parallel tracking of anatomy and 13C-Ala means

that Ala accumulation can be related to localized structures and

the developmental changes that occur within them; and finally,

individual caryopses can be subjected to post hoc analyses (e.g.,

MS-based detection of othermetabolites). There are a number of

possible applications for this NMR approach in the general area

of studying plant metabolism in vivo. For example, one can

equally provide 13C-Ala (instead of 13C-Suc or other substrates)

to study the routes through which Ala is taken up and distributed

within the plant, becomes degraded, or is incorporated into

proteins.

Ala Synthesis Is an in Vivo Marker for Hypoxia in Plants

Ala is the characteristic and major free amino acid accumulated

when plants are suffering from hypoxia, as shown here for barley

(Figure 2C) and as previously shown for Zea mays, Medicago

truncatula, and Arabidopsis thaliana (Good and Muench, 1993;

Muench and Good, 1994; Ricoult et al., 2005, 2006; Miyashita

et al., 2007). Ala synthesis relies on the reversible action of Ala-

AT, with the direction of catalysis being able to flip rapidly,

depending on the local conditions in planta (Good et al., 2007;

Beatty et al., 2009). Because Ala synthesis is responsive to envi-

ronmental variation (Wallace et al., 1984; Limami et al., 2008), its

localized accumulation can be considered as a marker for the

metabolic state. We have demonstrated here that 13C-Ala syn-

thesis occurs most readily in the central part of the endosperm,

which is the region of the endosperm in which the conditions are

the most hypoxic (Figures 2C, 3B, and 3C). When the starchy

endosperm is replaced by the nucellar projection, as occurs when

Jekyll is suppressed, the level of 13C-Ala synthesis is retained in the

most hypoxic region of the caryopsis, even though the pattern of

Suc delivery has been greatly altered. Thus, it appears that the

accumulation of Ala acts as amarker for localized hypoxia, at least

in the developing seed, mirroring the situation in animal cells,

where Ala synthesis is widely used for the diagnosis of hypoxia-

related disorders (Nyblom et al., 2004).

The 13C-Ala imaging described here has the potential to

identify which specific tissue is the most susceptible to hypoxic

constraint. As the assay is an in vivo one, it represents an

alternative to oxygen-sensitive microsensors, which inevitably

cause a degree of tissue damage during penetration and in some

situations are not usable at all (due to particular tissue features).

We suggest that the relationship drawn here between Ala accu-

mulation and hypoxia in the endosperm can quite possibly be

extended to diverse plant tissues, such as the roots/stem

phloem, in which both elevated Ala synthesis/accumulation

Figure 6. The Accumulation of Ala and Suc in Jekyll Downregulated

Plants.

(A) Cross section through the central part of the Jekyll downregulated

endosperm, showing the enlarged callus-like nucellar projection, which

invades the endosperm cavity. Cross section through the central part of

a wild-type endosperm shows normal starchy endosperm. Arrows

indicate the cuticular layer.

(B) A 13C/1H-MRI image of the endosperm, showing the localization of

Ala (middle panel) and Suc (bottom panel) within the same endosperm.

The accumulation of Ala is strongest in the central part of the endosperm

and is different from the distribution pattern of Suc.

3048 The Plant Cell

and reduced oxygen levels have been observed (Good and

Crosby, 1989).

Ala-AT Is a Key for Metabolic Adjustments in

Central Endosperm

In Arabidopsis cell suspension cultures, the raising of oxygen

availability increases flux throughout the metabolic network,

including Ala-AT (Williams et al., 2008). In barley endosperm, not

only the metabolic flux responds to local oxygen availability but

also the direction of enzyme catalysis (Ala-AT) can turn around.

This adds another level of complexity to the regulation of in vivo

metabolism. The FBAmodel of the barley caryopsis predicts high

Ala synthesis rates via Ala-AT in central endosperm that in turn

inhibits pyruvate accumulation (Figure 4), a metabolite which is

increasingly produced via glycolysis under hypoxia. In the mito-

chondria, the oxoglutarate produced by Ala-AT can be used by

oxoglutarate dehydrogenase and SuccCoA ligase to produce

ATP, which would compensate in part for the reduced supply of

respiratory ATP. Thismay help tomaintain a high level of ATP and

other energy-rich intermediates in inner endosperm regions

(Figure 3). In our model, subsequent reactions via both GDH

and MDH provide the major source of NAD+ regeneration (which

represents a critical step under O2 deficiency). Thus, achieving a

higher flux through Ala-AT represents an advantageous alterna-

tive under conditions where the operation of the usual TCA cycle

is inhibited. Similar conclusions were drawn from studies on

photosynthetically active Arabidopsis tissues (Lee et al., 2008)

and metabolic changes in response to waterlogging in Lotus

japonicus (Rocha et al., 2010). By removing pyruvate, the acti-

vation of ethanol/lactate fermentation is inhibited (Figure 4A), and

the stimulation of alternative oxidase activity can be avoided

(Oliver et al., 2008). Ala synthesis under endogenous oxygen

limitation is regarded as a means to maintain glycolysis and

energy provision. The caryopsis can improve its energy effi-

ciency/homeostasis and thereby enhance productivity and per-

formance (De Block and Van Lijsebettens, 2011).

While root-specific isoforms of Ala-AT and other homologs

expressed in vegetative tissues respond to oxygen supply (Good

and Crosby, 1989; Good and Muench, 1993; Miyashita et al.,

2007), the endosperm-specific one does not (V. Radchuk, per-

sonal communication). A plausible interpretation is that in the

endosperm, posttranslational regulation of Ala-AT activity is of

particular significance. This represents a rational strategy given

that the high energy costs of shifting protein abundance via

transcription/translation (Piques et al., 2009).

The flux maps were derived from a specific constraints-based

analysis and have provided several striking and/or unexpected

predictions of the consequences of hypoxia, which naturally will

need to be confirmed experimentally. First, the FBA suggests

that SDH is inactive under hypoxic conditions. SDH is involved in

both the TCA cycle and the respiratory chain and has been

shown to become inactivated under various conditions, includ-

ing O2 deficiency (reviewed in Sweetlove et al., 2010). Lowering

the activity of SDH will reduce the flux through the TCA cycle

(Araujo et al., 2011), producing a compensatory rise in the

glycolytic flux (the latter being part of the pseudohypoxic re-

sponse seen in animal tumor cells; King et al., 2006). The picture

is therefore one where glycolysis is upregulated in the central

region of the barley endosperm, where the supply of sugar is

more than adequate (Figure 2B), while that of oxygen is limited

(Figures 3B and 3C).

Second, the FBA predicts that barley caryopses, unlike roots

(Armstrong et al., 1994; Bailey-Serres and Voesenek, 2008;

Miyashita and Good, 2008), neither accumulate Gaba nor invoke

the Gaba shunt pathway. Our data demonstrated that the Gaba-T

enzyme activity was marginally induced by hypoxia (Figure 4A),

while the level of SSA (its reaction product) rose in the interior

(hypoxic) region of the endosperm (Figure 3). Nevertheless, the

Gaba-T Vmax remained very low (see Supplemental Table 1 on-

line), and the concentration of Gabawas not affected by variation

in the supply of oxygen (Figure 4A). Thus, it is hardly likely that the

Gaba shunt can carry any significant flux in the endosperm. This

might represent an endosperm-specific adaptation to the situ-

ation where oxygen deficiency is long-lasting rather than tran-

sient (Rolletschek et al., 2004). Third, the suggestion is that in the

interior (hypoxic) region of the endosperm, the assimilation of

ammonium operates via GDH rather than GS/GOGAT. GDH is

among the genes upregulated by hypoxia (Bailey-Serres and

Voesenek, 2008; Branco-Price et al., 2008), and its product

delivers a more energy efficient assimilation than GS/GOGAT

provides (Qiu et al., 2009). In the central hypoxic zone of the

endosperm, ammonia is used to generate Glu via GDH (Figure

5B), while in its more peripheral (oxygenated) regions, GDH does

not assimilate ammonia, but rather releases it. The measured

levels of Glu, Ala, and ammonia (Figure 3; see Supplemental

Table 1 online) are in line with this prediction.

Implications of the Existence of Metabolic

Compartmentation for Seed Filling

Our data demonstrate that the Suc transported to the caryopsis

moves via the well-oxygenated nucellar projection toward the

central endosperm (see Supplemental Movie 2 online), where an

appreciable level of hypoxia prevails. In central endosperm, a

significant level of localized Ala-AT activity is established, and

this synthesizes Ala (Figure 2C) to avoid pyruvate accumulation/

fermentation and to provide additional ATP. Eventually the

nitrogen use efficiency is also improved due to the reutilization

of ammonia, which is released by deterioration of adjacent

nucellar tissues (Radchuk et al., 2006). Multiple plasmodesmata

interconnect the cells (see Supplemental Figure 3 online), so that

the energy efficient movement of Ala along a concentration

gradient toward themore peripheral endosperm cells is enabled.

Since the peripheral cell is not in a hypoxic state, Ala-AT can act

to reconvert Ala back to pyruvate (Figure 4B).

The proposed metabolic compartmentation of the endosperm

is also mirrored by the structure of the tissue, since the central

part of the endosperm is composed largely of large cells,

whereas those at the periphery are small (see Supplemental

Figure 3A online). Although possibly coincidental, this pattern of

cell size corresponds to the known promoting effect of hypoxia

on cell expansion/elongation (Armstrong et al., 1994; Bailey-

Serres and Voesenek, 2008).

Hypoxia is commonly assumed to stunt vegetative growth and

compromisecellmetabolism (Armstronget al., 1994;Bailey-Serres

Compartmentation of Cereal Endosperm 3049

and Voesenek, 2008), but it now appears to be somewhat

beneficial in certain regions of the caryopsis. Constraints are

not uniformly experienced throughout a caryopsis, as a result of

the architecture of the organ and the nonuniform availability of

Suc/oxygen. These imbalances, as demonstrated by this exper-

imental and modeling approach, can be balanced by local

metabolic adjustments. We suggest that the metabolic com-

partmentation of the starchy endosperm provides a mechanism

to ensure metabolic flexibility and eventually contributes to the

high carbon conversion efficiency shown by the cereal starchy

endosperm (Alonso et al., 2011).

METHODS

Plant Growth and Stable Isotope Labeling

Barley plants (Hordeum vulgare cv Barke and Golden Promise) and

transgenic Jekyll downregulated barley plants were grown under stan-

dard greenhouse conditions at 188C with 16 h of light and a relative air

humidity of 60%. Determination of developmental stages for developing

barley caryopses and tissue isolations were performed as described by

Radchuk et al. (2006). Stable isotope labeling was performed using

previously used 13C feeding procedures (Singh and Jenner, 1983; Ekman

et al., 2008). Stems were cut 5 cm below the ear and placed in nutrient

solution containing quarter-strength Murashige and Skoog medium, 10

mM Gln, 10 mM Asn, 2 mM MES buffer, pH 6.0, and 100 mM UL-13C12-

Suc (Omicron Biochemicals).

NMR Experiments

The NMR experiments were performed on a 17.6 T wide bore (89 mm)

superconducting magnet (Bruker BioSpin) with a maximum gradient

strength of 1 mT m21 using a custom built double resonant 13C/1H-NMR

coil (inner diameter 5 mm).

For metabolic 13C imaging, the geHMQC pulse sequence with phase

encoding gradients for spatial localization was used (see Supplemental

Figure 2A online). An additional frequency selective water saturation

scheme (VAPOR; Tkac et al., 1999) was built into the sequence for

efficient suppressing of the water signal. The geHMQC sequence con-

sisted of a slice 908 selective excitation pulse on the protons, followed by

phase encoding gradients in the remaining two spatial dimensions. After

t = 1/(2 1JCH), a 908 hard pulse was applied on 13C channel, which creates

multiple quantum coherences (MQCs). The following 1808 pulse on the

protons interchanges the MQCs and refocuses the chemical shift evolu-

tion. The last 908 pulse on 13C converts MQCs back to detectable

magnetization. TheMQC gradients G1, G2, andG3were used in a ratio of

2:2:1 to select a coherence pathway and to suppress signal from protons,

which are connected to 12C. This MQC gradient combination allows

refocusing 50%of the 1Hmagnetization coupled to 13C spins. The spoiler

gradients (Sp) were used to dephase magnetization, which results from

imperfections of the 1808 pulse. A total of 2048 complex points were

acquired at a bandwidth of 20 kHz with 13C decoupling (Malcolm Levitt

decoupling), which suppresses the 1JCH coupling and improves the SNR.

Further experiment parameters are as follows: TR = 1.5 s, TE = 8.2ms, t =

2.9 ms, number of scans = 800 (hanning-weighted k-space acquisition

scheme), FOV = 8 3 8 mm2, spatial resolution 0.4 3 0.4 mm2, slice

thickness = 2.5 mm, MQC gradient strength (G1: G2: G3) = (0.4: 0.4: -0.2)

T/m, MQC gradient duration = 1 ms, water suppression bandwidth = 500

Hz, duration for one experiment = 20 min.

Structural 1H imaging was performed using a multislice spin echo

sequence: TR = 5 s, TE = 5.1 ms, FOV = 20 mm, Matrix = 192 3 192,

resolution = 423 42mm2, slice thickness = 200mm, number of slices = 55,

averages = 1, experiment duration = 16 min.

Image reconstruction and analysis was done with in-house written

software (Java and MATLAB 7.9.0; The MathWorks). The data were

filtered with an exponential function (time constant = 3.3 ms) in the time

domain to improve the SNR. For the dynamic uptake study of 13C-Suc,

the data for every frame were averaged from seven consecutive exper-

iments. The data from these frames were linear interpolated to create

additional frames for a smoother video (see Supplemental Movie 2

online).

Experimental Treatment of Samples Used for Biochemical Analysis

To study the effect of distinct light and oxygen regimes on metabolism of

developing caryopses (12 to 16 d after fertilization), intact ears were kept

in either light (600 mE) or darkness (enclosed with black paper) for 6 h. In

addition, some dark-incubated ears were aerated with gas mixtures

containing either reduced (2.1 kPa) or elevated oxygen levels (42 kPa;

balanced by N2 via a multigas controller). After 6 h, caryopses were

quickly removed and immediately frozen in liquid nitrogen. To analyze

metabolite pattern in inner versus outer regions of endosperm, freshly

harvested caryopses were snap-frozen in liquid N2 and freeze-dried.

Subsequently, the outer pericarp layers were peeled off and the endo-

sperm was separated into inner and outer parts by use of a razor blade.

Metabolite Extraction and Analysis

For analysis of 12/13C-Ala and sugars, frozen caryopses were homoge-

nized in 2-mL microcentrifuge tubes (Eppendorf) containing two steel

beads (ASK) by grinding in a Retsch TissueLyser (Qiagen) for 45 s at 1800

strokes21. After homogenization, the sampleswere extractedwith 0.5mL

1:1 (v/v)methanol/water under the samegrinding conditions. The extracts

were filtered with Vivaclear centrifugal filters (0.8 mm pore size; Satorius-

Stedim Biotech) at 2000g for 2 min. Samples were analyzed in an LC-MS/

MS system (Dionex Ulimate 3000 RSLC and API 4000 [Applied Biosys-

tems]). Onemicroliter was injected onto a LUNA-NH2 (5mm, 1503 2mm;

Phenomenex) attached to a precolumn (4 3 2 mm; Phenomenex). As

mobile phases acetonitrile (solvent A) and water (solvent B) were used

with a flow of 0.4mL/min in a gradientmodewith the following conditions:

time/concentration (min/%) for B: 0.0/10, 1.0/10, 2.0/55, 3.5/55, 3.7/10,

and 5.0/10. The mass spectrometer was used in the ion negative mode,

and ions were detected using multiple reaction monitoring. The following

transitions were observed: substance/Q1 mass/Q2 mass/declustering

potential/collision energy for 12C3-Ala/88/88/-33/-5, for 13C3-Ala/91/91/-

33/-5, for Suc/341/89/-90/-25, for 13C12-Suc/353/92/-90/-25, for fruc-

tose/179/89/-35/-12, for 13C6-fructose/185/92/-35/-12, for glucose/179/

89/-35/-12, and for 13C6-glucose/185/92/-35/-12. Nitrogenwas used as a

curtain gas, nebulizer gas, heater gas, and collision gas. The ion spray

voltage was set to24200 V, the capillary temperature was 3008C, and the

dwell time for all compounds was 80 ms.

For analysis of other metabolites, frozen caryopses were grinded as

above and extractedwith chloroform/methanol/water (1:1:3) according to

Soga et al. (2002). The analysis of soluble sugars was performed by ion

chromatography using pulsed amperometric detection (Rolletschek

et al., 2005). Ammonia was measured by ion chromatography coupled

to conductivity detection (ICS-3000; Dionex); separation was done using

the IonPacCS12A (43 250mm) in the isocraticmodewith 10mMH2SO4.

The remaining metabolites were measured by LC-MS using three

different methods. Ion chromatography (ICS-3000) coupled to an electro-

spray ionization triple quadrupole mass spectrometer (API 4000) in

negative mode was used for compounds listed in Supplemental Table 2

online. The separation was performed on an IonSwift MAX-100 (1 3 250

mm; Dionex) with a constant column temperature of 408C and a column

3050 The Plant Cell

flow of 150 mL/min. The suppressor was set to a value of 38 mV. As

sodiumhydroxide eluent, we used the following gradient: t = 0min (5mM);

t = 10 min (5 mM); t = 16 min (12 mM); t = 28 min (25 mM); t =32 min (100

mM); t = 38 min (100 mM); t = 42 min (5 mM); t = 56 min (5 mM).

The other methods were performed with the coupling of LC (Ultimate

3000) and the same mass spectrometer. The hydrophilic interaction

chromatography method with an aminopropyl column (Phenomenex

Luna NH2; 250 mm 3 2 mm, particle size 5 mm) is based on Bajad

et al. (2006). To obtain maximum separation of different compounds, the

column was used in combination with the mass spectrometer in negative

and positive mode and varying eluent gradients. The LC eluents are as

follows: solvent A, 20 mM ammonium acetate + 20 mM ammonium

hydroxide in 95:5 water:acetonitrile, pH 9.45; solvent B, acetonitrile. The

gradients in the negative mode to determine the metabolites (listed in

Supplemental Table 3 online) are as follows: t = 0, 85% B; t = 15 min, 0%

B; t = 38min, 0%B; t = 40min, 85%B; t = 50min, 85%B. The gradients in

the positive mode to determine the metabolites (listed in Supplemental

Table 4 online) are as follows: t = 0, 85% B; t = 15 min, 0% B; t = 28 min,

0%B; t = 30min, 85%B; t = 40min, 85%B.Datawere normalized to plant

milligrams of fresh weight and to an internal standard added during

extraction (13C-succinate; Cambridge Isotope Labs).

Determination of Ethanol

Samples were ground with mortar and pestle, and 1000 mL of methanol

(100%,MS grade) was added. After shaking for 1 h at 108C, samples were

cleaned using Vivaclear filters and filled into gas chromatography vials.

Ethanol concentration in the extracts was measured by a gas chromato-

graph (model 2014; Shimadzu) equipped with a flame ionization detector.

The column was Zebron ZB-1 with dimensions 3 mm, 60-m length, and

0.32-mm inner diameter. The following analysis parameters were used:

injector temperature, 2008C; carrier gas, He; linear velocity, 35 cm/s;

split ratio, 30; oven temperature program, 508C hold for 5min, increase to

708C within 2min, followed by temperature rise to 2408C at 508C/min;

detector temperature, 2508C. Limit of detection was determined as

;1 nmol ethanol/g freshweight (based on additions of known standards).

FBA

FBAwas applied to a recently establishedmodel of primarymetabolism in

the barley caryopsis (Grafahrend-Belau et al., 2009a). To elucidate the

role of Ala accumulation during hypoxia, the model was extended by

reactions involved in hypoxic metabolism (i.e., Ala export, mitochondrial

Ala-AT, export of TCA cycle intermediates, and Gaba export). Assuming

that efficient use of energy is the main objective of hypoxic metabolism,

theminimization of ATP production per flux unit was used as the objective

function for the hypoxic growth simulations. The objective was computed

as a two-step optimization process, where the first step is to minimize

ATPproduction and the second step is tominimize the overall intracellular

flux. The biological assumption underlying the objective function is that

the cells grow while (1) using the minimal amount of energy, resulting in

energy conservation (first optimization step), and (2) achieving an efficient

channeling of metabolites (second optimization step). The computation

was performed by fixing the growth rate at 1.8 h21, a value representative

for the central endosperm, and by adding the objective value of the first

optimization as an additional constraint to the second optimization. With

respect to the aerobic growth simulations, the maximization of biomass

per flux unit was used as the objective function (Grafahrend-Belau et al.,

2009a).

All simulations were performed by constraining the uptake rate for Suc

between 0 and 8mmol g21 DWh21, a value experimentally determined for

the developing grain of barley (Felker et al., 1984). Due to the lack of

barley-specific data, the uptake rate for Asp and Glu was constrained

between 0 and 4 mmol g21 DW h21, based on experimental reports of

soybean (Glycine max) seeds (Iyer et al., 2008; Allen et al., 2009,). In

addition, Ala was allowed to be taken up under aerobic growth conditions

by constraining the exchange flux between 0 and 6 mmol g21 DW h21; the

latter value corresponds to themaximal hypoxic Ala accumulation rate. In

accordance with the experimental results, the accumulation of the

classical fermentation products (ethanol and lactate) was inhibited (eth-

anol) or restricted to aminimal value (lactate, 0.4 mmol g21 DW h21) under

hypoxic growth conditions. To simulate growth under different O2 supply,

the O2 uptake rate was fixed at representative values for each tissue

(central endosperm, 3mmol g21 DWh21; peripheral endosperm/pericarp,

10 mmol g21 DW h21). The latter value corresponds to respiration rates

measured for whole caryopses (ranging between 10 and 13 mmol oxygen

g21 DWh21 at the stage analyzed here; Tschiersch et al., 2011). The same

biomass composition for both the central and the peripheral endosperm

was used for modeling because starch is the major storage product all

over the endosperm tissue at the stage analyzed here (Radchuk et al.,

2009) and accumulation of lipid and protein in aleurone occurs later in

development (Neuberger et al., 2008).

To elucidate the in silico function of Ala-AT in response to varying O2

supply, simulations were performed with the Suc uptake rate fixed at 8

mmol g DW21 h21, the O2 uptake rate varying from anaerobic to aerobic

conditions (1 to 10 mmol g DW21 h21) and the maximization of biomass

per flux unit chosen as the objective function. The remaining exchange

fluxes were constrained as described above. Simulations as well as

visualization of the resulting fluxmapswere performed using FBA-SimVis

(Grafahrend-Belau et al., 2009b).

Enzyme Extraction and Analysis

Extraction and activity assays for enzymes were done according to

previously described protocols: Ala-AT (EC 2.6.1.2; Good and Crosby,

1989), Asp-AT (EC 2.6.1.1; Griffith and Vance, 1989), g-aminobutyric acid

aminotransferase (EC 2.6.1.19; Miyashita and Good, 2008), pyruvate

decarboxylase (EC 4.1.1.17; Rolletschek et al., 2002), alcohol dehydro-

genase (EC 1.1.1.1; Rolletschek et al., 2002), lactate dehydrogenase (EC

1.1.1.27; Rolletschek et al., 2002), and pyruvate kinase (EC 2.7.1.40;

Rolletschek et al., 2005). The coupled spectrophotometric assays were

tested for substrate dependence and linearity with time and amount of

extract.

Generation of Oxygen Maps, Microsensor Analysis, and

Micro-Computer Tomography

Oxygen maps are based on O2 microsensors studies (Rolletschek et al.,

2004). Spatially highly resolved O2 concentration profiles were derived

frommicrosensor insertions along various transects across the caryopsis

and drawn as isolines of O2 concentration. The oxygen influx was

calculated from the gradient in oxygen concentration across the corre-

sponding region of endosperm. Oxygen measurements in Jekyll plants

were done according to Rolletschek et al. (2004).

Structural models of barley caryopsis (12 d after flowering) were

acquired by applying the manufacturer’s protocols and a Skyscan1172

instrument.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Characteristics of the Barley Starchy Endo-

sperm during Grain Filling.

Supplemental Figure 2. The geHMQC Pulse Sequence and in Vitro

Spectra.

Compartmentation of Cereal Endosperm 3051

Supplemental Figure 3. Structural Characteristics of Starchy Endo-

sperm.

Supplemental Table 1. Steady State Metabolite Levels and Enzyme

Activites (Vmax) in Endosperm Developing under Ambient and Ele-

vated Levels of Oxygen Supply.

Supplemental Table 2. Parameters for Mass Spectrometric Analysis

of Metabolic Intermediates Coupled to Ion Chromatography.

Supplemental Table 3. Parameters for Mass Spectrometric Analysis

of Metabolic Intermediates Coupled to Hydrophilic Interaction Chro-

matography (Negative Mode).

Supplemental Table 4. Parameters for Mass Spectrometric Analysis

of Metabolic Intermediates Coupled to Hydrophilic Interaction Chro-

matography (Positive Mode).

Supplemental Data Set 1. Steady State Metabolite Levels in the

Central and Peripheral Regions of the Barley Endosperm, as Mea-

sured by LC-MS.

Supplemental Data Set 2. Simulated Flux Values in the Central and

Peripheral Regions of the Endosperm.

Supplemental Movie 1. A Three-Dimensional Reconstruction of the

Barley Ear, as Measured by NMR.

Supplemental Movie 2. The Allocation of 13C-Suc inside the Barley

Caryopsis, as Measured by NMR.

ACKNOWLEDGMENTS

We thank K. Blaschek for excellent technical assistance. This research

was financially supported by the German Federal Ministry of Education

and Research (GABI project “SysSeed”).

AUTHOR CONTRIBUTIONS

H.R. and L.B. designed the research, performed research, analyzed data,

and wrote the article. E.G.-B. and N.H. performed the research. G.M.,

J.F., and P.M.J. contributed new analytic tools (NMR). F.S. and P.M.J.

analyzed data.

Received May 4, 2011; revised July 1, 2011; accepted July 31, 2011;

published August 19, 2011.

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3054 The Plant Cell

DOI 10.1105/tpc.111.087015; originally published online August 19, 2011; 2011;23;3041-3054Plant Cell

Schreiber, Peter M. Jakob and Ljudmilla BorisjukHardy Rolletschek, Gerd Melkus, Eva Grafahrend-Belau, Johannes Fuchs, Nicolas Heinzel, Falk

in the Barley EndospermCombined Noninvasive Imaging and Modeling Approaches Reveal Metabolic Compartmentation

 This information is current as of October 26, 2020

 

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