Download Positional cues for the starch/lipid balance in maize kernels and

The Plant Journal (2005) 42, 69–83
doi: 10.1111/j.1365-313X.2005.02352.x
Positional cues for the starch/lipid balance in maize kernels
and resource partitioning to the embryo
Hardy Rolletschek1, Karen Koch2, Ulrich Wobus1 and Ljudmilla Borisjuk1,*
Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, 06466 Gatersleben, Germany, and
2
Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, 1143 Fifield Hall, PO
Box 110690, Gainesville, FL 32611, USA
1
Received 22 October 2004; revised 17 December 2004; accepted 23 December 2004.
*
For correspondence (fax þ49 39482 5500; e-mail [email protected]).
Summary
This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect
assimilate partitioning and ATP distribution within the kernel. These questions were addressed through
combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis.
Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2
concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation
(approximately 3.8 lM), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact
kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP
ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the
citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch
formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low
oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability
increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation
into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with
labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded
ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be
responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that
the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors
assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for
peripheral localization of the protein and lipid storing structures in maize kernels.
Keywords: lipid and starch metabolism, metabolite imaging, seed development, assimilate partitioning,
energy state, hypoxia.
Introduction
Maize is one of the world’s top three grain crops (Chrispeels
and Sadava, 2003), with starch, lipid, and protein deposition
in this kernel accounting for a significant portion of the
global food supply. Its endosperm is the major site of starch
storage, whereas lipid biosynthesis is favored in its embryo
(80 and 30%, respectively; Doehlert, 1990). Extensive study
of assimilate partitioning between these seed structures and
storage components has shown their regulation to involve a
complex interplay of gene expression and metabolism during development (Bate et al., 2004; Becraft, 2001; Choi et al.,
ª 2005 Blackwell Publishing Ltd
1997; Focks and Benning, 1998; Hannah and Greene, 1998;
Lin et al., 1999; Sakulsingharoj et al., 2004; Wobus and
Weber, 1999).
Kernel development is supported by sucrose transferred
from phloem, across pedicel and basal transfer cells (Aoki
et al., 1999; Thomas et al., 1992) to the main endosperm
compartments and embryo (Shannon, 1972). Sucrose is
partly cleaved and re-synthesized within the endosperm
(Cheng, 1997), which in turn might affect developmental state
(Wobus et al., 2004) and expression of storage-associated
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70 Hardy Rolletschek et al.
genes (Koch, 1996, 2004). Sugars subsequently move toward
the upper endosperm where starch storage is initiated. Flux
toward the embryo seems to be tightly controlled by specific
features of the embryo-surrounding tissue, responsible
mechanisms being largely unknown, but probably involving
invertases and their inhibitors (Bate et al., 2004). Starch
biosynthesis in endosperm often correlates with activity of
sucrose synthase (Doehlert, 1990; Winter and Huber, 2000)
and is typically limited by ADP-glucose pyrophosphorylase
activity (Hannah and Greene, 1998; Salamone et al., 2002).
Lipid biosynthesis in the embryo seems more closely associated with invertase and hexokinase activity (Doehlert,
1990). Enzymes of glycolysis, citrate cycle, starch, protein
and lipid biosynthesis are reported to be coordinately
expressed in both endosperm and embryo (Giroux et al.,
1994; Lee et al., 2002). Their activity determines local sink
strength which in turn may regulate assimilate partitioning
into different storage product classes.
Recent data suggest that low internal oxygen concentrations may also play a pivotal role in assimilate
partitioning. Oxygen depletion appears to be a common
feature of many plant tissues, including developing seeds
and grains (Rolletschek et al., 2002a; Shelp et al., 1995;
Vigeolas et al., 2003). Endogenous hypoxia was shown to
decrease assimilate import and storage metabolism by
wheat grains (VanDongen et al., 2004), and to alter energy
state of growing barley kernels (Rolletschek et al., 2004a).
Analysis of diverse dicot seeds also showed that endogenous oxygen limitation affected resource allocation
among storage products (Borisjuk et al., 2003; Rolletschek
et al., 2003). In embryos of the dicot, oilseed rape, Vigeolas
et al. (2003) could show that lipid but not starch storage
metabolism was limited by the prevailing low oxygen
levels. Photosynthesis was also shown to provide O2 to
hypoxic seed tissues (Rolletschek et al., 2004a) as well as
energy (ATP/NADPH; Ruuska et al., 2004). There are several reasons why developing maize kernels may be subject
to still greater effects of low oxygen than the dicot seeds
and smaller grains. One is that the surface/volume ratio of
the large maize kernels would be smaller. In addition,
maize kernels are distinct in (i) their virtually complete lack
of photosynthetic activity in the grain, and (ii) the extensive non-vascular region (no lateral vein supplies the
grain). These factors indicate that endogenous oxygen
deprivation may be particularly pronounced inside developing maize kernels, and that evolution of this largest
cereal grain may have involved special adaptations to
oxygen limitation.
Significant implications of oxygen depletion (in sensu
energy limitation) could include altered input into signaling systems, gene expression, and/or metabolic control
(Geigenberger, 2003; Greenway and Gibbs, 2003; Koch
et al., 2000; Subbaiah and Sachs, 2001). Both transport
and phosphorylation of hexoses via hexokinases are
affected by low O2/ATP (Bouny and Saglio, 1996;
Geigenberger, 2003; Trethewey et al., 1999; Xia and Saglio,
1990). In addition, maize sucrose synthases (Sus1 and Sh1)
respond differentially to both sucrose and low oxygen
(Zeng et al., 1998). Moreover, the balance between activity
of the sucrose-cleaving enzymes, invertase and sucrose
synthase, is affected by low O2 (Zeng et al., 1999). This in
turn would be expected to alter the hexose/sucrose state of
the cells, and partitioning of C-flux between storage and
respiration.
The overall motivation for this work was to investigate the
extent of potential oxygen depletion in functionally different
regions of maize kernels, and determine the degree to which
this limits key aspects of kernel metabolism: assimilate
partitioning and C-flow to the embryo. Toward this end, we
combined topographical analysis of ATP gradients across
tissues in developing maize kernels, with microsensor
quantifications of O2 in transects through kernels, and
assays of energy balance, metabolite profiles, and labeling
studies with 13C- and 14C-assimilates. Each approach was
applied to experimental perturbations of oxygen inside
intact, attached kernels. Results show a marked degree of
oxygen deficiency inside developing maize kernels. Moreover, data reveal that young kernels contend with significant
oxygen limitation to ATP gradients, energy status, metabolite pools, assimilate delivery to developing embryos, and
partitioning of sucrose to non-starch storage compounds.
Starch storage in endosperm, however, appears specifically
adapted to the localized low-oxygen environment inside
these, and possibly other grains.
Results
Oxygen is severely depleted inside developing maize
kernels
Experiments were performed during the near-linear phase of
starch deposition in developing maize kernels (from about
100 to 350 mg fresh weight, approximately 12–42 days after
pollination, DAP). Onset of this stage was marked by
increased levels of ADP-glucose (the direct precursor for
starch synthesis) (Figure 1a). Amounts of glycolytic intermediates did not change significantly during this period, but
free amino acids generally declined (data not shown). The
ratio of whole-kernel ATP to ADP remained relatively constant, but decreased somewhat during the storage phase
(Figure 1b), consistent with overall increases in ATP demand
versus ATP supply. A 10-fold decrease in the kernel hexose/
sucrose ratio was observed at the onset of starch storage
(9.8 versus 0.8). However, hexose levels were considerably
lower in embryos than in the (mainly) starch-storing endosperm (Figure 1c), indicating a distinctly different metabolic
status. Such depletion of hexoses relative to sucrose in
embryos could readily alter sugar signals in these structures,
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 71
larger kernels. In contrast to endosperm, embryos showed
two remarkable features: (i) Mean O2 levels remained
considerably higher (at about 13.5 6.5%). (ii) Concentration gradients descended from relatively high levels at the
outer surface of the embryo (10–20%) to barely detectable
amounts at the embryo/endosperm interface (innermost
scutellar surface). This concentration gradient suggests that
diffusive influx through the embryo may be an important
source of oxygen for the scutellum and the embryosurrounding region.
ATP is gradiently distributed within the kernel tissues
Figure 1. Biochemical parameters of developing maize kernels.
(a) Dynamic of starch accumulation (mg per kernel) and ADP-glucose levels
(nmol g)1 fresh weight).
(b) Decrease of ATP/ADP-ratio during starch storage.
(c) Contrast between mean sugar status of embryo and endosperm (25 DAP).
Error bars are mean standard deviation.
and thus differentially affect developmental programs (Bate
et al., 2004; Wobus and Weber, 1999).
Internal oxygen levels were quantified in young kernels
using microsensors with tip diameters of approximately
50 lm, which allowed a high degree of spatial resolution
(Figure 2). The O2 level dropped dramatically within the first
500 lm of the kernel surface. The remainder of the endosperm showed little detectable O2, with mean levels in the
interior reaching only 1.4 1.0% saturation (atmospheric O2
level of approximately 21 kPa is set to 100%). Minimum
levels dropped below resolution of the microsensor (<0.1%).
As the microsensor tip moved through the kernel and
approached the pericarp surface on the opposite side, O2
levels again increased steeply and nearly symmetrically. The
mean O2 levels within the endosperm were equally
depressed in kernels from 12 to 45 DAP, but were considerably higher in kernels prior to onset of starch storage
(<10 DAP; data not shown). Measurements of O2 levels
within the embryo alone were technically not feasible for
kernels smaller than 200 mg, but were reproducible for
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
We further analyzed spatial ATP distribution and whether
gradients in ATP are related to those in local oxygen levels
and storage-product deposition. ATP distribution was
determined directly in cryosections of snap-frozen kernels
via bioluminescence imaging (Borisjuk et al., 2003). Data
from whole kernels sectioned as in Figure 3(a), and shown in
Figure 3(b,d,e), reveal that ATP levels were low in peripheral
regions of kernels (pericarp, pedicel, aleurone, and outermost endosperm). ATP levels remained high throughout
most of the inner endosperm, with steep concentration
gradients at the periphery (Figure 3c). ATP content increased
almost linearly from peripheral endosperm regions with
small cells toward inner regions with larger cells. More
gradual ATP gradients were evident between basal and
apical parts of the endosperm.
Transverse sections along the central a-axis (Figure 3d)
also showed that ATP levels were low in peripheral regions
of the kernel, but higher in conducting tissues. Greatest ATP
levels (up to 1000 nmol g)1 FW) were observed in the
endosperm region immediately adjacent to the embryo
base. ATP concentration dropped markedly in the lower
endosperm, particularly in the gap region, the transfer cells,
and in adjacent tissues. Content of ATP was lower in the
embryo axis than in the endosperm, but in scutellum, an
ATP gradient extended from the abaxial epidermis toward
the inner zone, peaking in the abaxial nodular region.
Scutellar procambium and inner tissues of the embryo
(coleoptile, plumule, primary root and others) showed low
ATP levels. Within maternal tissues (shown along the c-axis
in Figure 3e), ATP levels were slightly elevated in the spongy
parenchyma, but very low in the vascular bundle. ATP
content was also low at the endosperm/pericarp interface.
A comparison of Figure 3(c,f) shows that ATP levels were
generally higher in large cells of the interior endosperm and
embryo-surrounding regions than in more dense tissues
with small cells. For each region of ATP analysis (Figure 3c),
tissue masks in gray scales (base of Figure 3c) delineated
distribution of small cells in peripheral endosperm and
larger cells in loose tissues of the central region (as pictured
in Figure 3f). The layers of endosperm cells lining the
embryo pocket also contained larger cells than the outer
72 Hardy Rolletschek et al.
endosperm layers away from the embryo (not shown). The
described pattern is in agreement with a model that quantitatively reproduced the cell-size gradient within maize
endosperm (Vilhar et al., 2002). Similar cell-size gradients
within the endosperm have been described in detail for
different maize lines (Kowles and Phillips, 1988; Larkins
et al., 2001). Superimposition of our ATP maps with such
cell-size gradients revealed the general association of ATP
content and cell size in the endosperm.
In addition, parallel histological analysis showed that
although starch was present throughout the upper half of
endosperm (Figure 4a,b), gradients were evident in density
(Figure 4b) and size (Figure 4c,d) of starch grains. The
density decreased basipetally in both central (Figure 4b)
and lateral regions (not shown). Differences in size of cells
and starch grains were most clearly apparent in comparisons of peripheral and central endosperm (Figure 4c,d,
respectively). Large central cells held the biggest starch
grains (with lowest surface-to-volume ratios). In embryo,
only small starch grains were observed, mostly located in
scutellum. Maternal tissues were filled with starch as
well. The pattern described here is consistent with earlier
investigations of starch deposition and gene expression
(Perdomo and Burris, 1998; Young et al., 1997). Lipid staining with Sudan B red showed that lipids accumulated mainly
in the embryo and in the aleurone cells as well as in the
endospermal cells adjacent to the aleurone layer (Figure 4e).
Only residual levels of lipids were detectable in the central
endosperm or embryo-attached regions. Strong lipid gradients were visualized within the embryo (stained image in
Figure 4f). Lipids accumulated mainly in the region facing
the endosperm and decreased toward the embryo interior.
Similar spatial and temporal patterns were observed previously for expression of the L3 lipid body protein gene (Vance
and Huang, 1988). Notably, the present work shows that lipid
gradients within the embryo were coupled with ATP, i.e.
lipid deposition decreased concomitant with ATP levels
(Figure 4f).
A comparison of the ATP gradient maps to both storage
pattern and oxygen distribution within the kernel supported
the following observations: (i) In endosperm, where starch is
the main storage product, oxygen concentration was very
low and high ATP levels were associated with enlarged,
starch-storing cells. (ii) In the embryo, where lipids are the
main storage product, oxygen levels were relatively high
and gradients of ATP coincided spatially with those of lipids.
This implies that high ATP levels may be related to specific
storage activities in different regions of the seed.
Figure 2. Representative oxygen maps within developing maize kernels. O2
was measured by microsensors along the x-axis (penetration depth given in
lm) within kernels at (a) 130 mg fresh weight (15 DAP), (b) 230 mg fresh
weight (25 DAP), and (c) 300 mg fresh weight (35 DAP). The O2 concentration
is given in % of atmospheric saturation (approximately 21 kPa ¼ 100%).
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 73
Figure 3. Bioluminescence imaging of ATP distribution within maize kernels during the peak
period of starch storage.
(a) Orientation of a, b, and v sections through the
maize kernel. ATP concentrations are given in a
color scale at the base (en, endosperm; e,
embryo; p, pericarp).
(b, d, e) Left panels represent ATP maps (gradients and their physical localization) measured
directly within cryosections positioned as shown
for a, b, and v sections in the right panels (BE,
basal endosperm; CE, central endosperm; P,
pericarp; PC, pedicel; PE, peripheral endosperm;
Sc, scutellum; VB, vascular bundle).
(c) Graph of ATP concentration measured across
the dashed arrow in the right panel of (b) is
pictured in relation to (f) a co-localized gradient
in cell size with iodine staining of amyloplasts in
this starchy endosperm tissue. Cell conture is
marked by the white line to visualize the cell-size
gradient.
Figure 4. Distribution of storage products in
longitudinal sections of kernels during the peak
period of starch storage.
(a) Hand-section through the kernel.
(b) Starch deposition visualized in dark-brown by
iodine staining.
(c, d) Comparison of starchy endosperm cells
from peripheral (c) and central (d) endosperm.
(e) Lipid accumulation visualized in red by
Sudan B.
(f) Lipid deposition within embryo in relation to
concentration gradients for ATP (line graph) and
oxygen (circles). Both ATP and O2 levels were
measured along a transect line (white arrow) also
shown as the x-axis in panel (f). The ATP line
graph represents a polynomial regression curve
fitted to individual ATP analyses (black dots) (Al,
aleurone; G, germ; Pc, pedicel; e, embryo; en,
endosperm; Sc, scutellum).
External O2 concentration affects internal O2 levels, energy
state and ATP distribution
As shown above, O2 levels within kernels fall to very low
levels. To determine whether the interior of developing
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
maize seeds are hypoxic under in vivo conditions, we
experimentally altered the exogenous O2 supply to kernels
(Figure 5). The microsensor was inserted into intact, attached kernels (about 350 lm depth), followed by a stepwise
change of the ambient O2 levels. Reduction of external O2
74 Hardy Rolletschek et al.
(Figure 6a,b). The increase in energy levels resulting from
a rise in oxygen levels from the 10 to 200% level was more
marked for older than younger kernels.
To see if changes in the ATP level affected the whole
kernel or (even more importantly) were confined to distinct
regions otherwise concealed by whole organ analysis, we
investigated the ATP distribution using bioluminescence
imaging. When kernels were exposed to 10% O2, ATP
concentration decreased most markedly in the basal and
middle endosperm region near the embryo (Figure 6c).
However, when exogenous O2 was raised to 200% ATP-rich
regions began expanding into upper parts of the endosperm, the embryo surrounding region and the embryo
itself. In later stages of kernel growth (Figure 6d), the
decrease in ATP at the 10%-level, as well as the increase at
the 200%-level, was primarily associated with the embryo
and the embryo-surrounding region. These results imply
that an increase in O2 supply leads to higher energy levels
and altered ATP distribution patterns. Data also indicate that
ATP production in developing kernels is limited in vivo by
low internal oxygen levels (hypoxia).
Oxygen availability alters steady-state levels of metabolites
and enzyme activities
Figure 5. Effect of elevations in external oxygen levels on internal O2
concentration in the central endosperm of the maize kernel (25 DAP). The
level of O2 is given in % of atmospheric saturation (approximately
21 kPa ¼ 100%; mean standard deviation).
availability drove internal concentrations below levels of
detection within 3–5 min. Increases in external O2 concentration, up to approximately 200% (representing
42 vol.% ¼ twofold atmospheric O2 saturation), did not affect the internal level (Figure 5). More marked elevations of
external O2 concentration eventually raised endosperm
levels above those normally observed in vivo. Rising O2
supply was thus apparently balanced by increasing O2 consumption. External levels greater than 200% were needed
before internal O2 demand could be exceeded. Consequently, respiration inside developing kernels was considered to be non-O2-saturated at normal ambient levels, i.e.
was O2-limited in vivo.
To test if changes in respiratory O2 demand induced by
varying external O2 supply resulted in elevated energy
levels, intact kernels were treated with gas mixtures containing either 10, 100, or 200% of the ambient O2 level
(corresponding to 2.1, 21, and 42 vol.% of O2, respectively).
The concentration of adenine nucleotides in whole kernel
extracts increased progressively with increasing external
O2 levels (Figure 6a,b). The ATP/ADP ratio also increased
significantly in response to elevated external O2
Differential responses to changes in O2 supplies are shown
in Figure 7 (see also supplementary Materials Table S1) for
intermediates of glycolysis and the citric acid cycle, as well
as for related sugars and free amino acids. The positioning
of sub-figures is based on their proximity in a generalized
metabolic context (for details see Dwyer et al., 2004).
Although soluble sugar levels did not change in response to
shifts in external O2 supply, UDP-glucose content dropped
markedly when oxygen availability was doubled. Sucrose
synthase activity also declined from 2.8 (0.2) to 2.0 (0.4)
lmol g)1 FW min)1. In contrast, no significant change was
observed for either the ADP-glucose pool size or activity of
ADP-glucose pyrophosphorylase [2.4 (0.3) lmol g)1
FW min)1]. Levels of most glycolytic intermediates rose
progressively with increasing O2 supply, but those of pyruvate were maximal when exogenous O2 supplies (10%) were
lowest. The lack of accompanying increases in either lactate
or alanine, indicated an absence of lactate/alanine fermentation even under these low-O2 conditions. Metabolite levels
that increased most markedly in response to superambient
oxygen were Fruc1,6diP and acetyl-coenzyme A, the latter
being a precursor for plastidic fatty acid synthesis as well as
mitochondrial citrate cycle, amino acid synthesis, etc. Within
the pool of organic acids, only citrate and isocitrate showed
significant, progressive increases with rising O2 availability,
and similar patterns were evident for Glu, Asp, Ser, and
some of the other free amino acids [Gln, Gly, Pro (not
shown)]. Together, observed changes showed that increases in O2 levels allowed supplies of glycolytic intermediates
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 75
Figure 6. Effect of changing external oxygen
levels on concentration of adenine nucleotides,
ATP/ADP ratio and ATP distribution in maize
kernels at 27 DAP (a, c) and 42 DAP (b, d). Intact
kernels were aerated with gas mixtures containing 10, 100 and 200% of ambient oxygen levels
(black, gray, and white columns, respectively).
Data in (a) and (b) are given as mean standard
deviation. Concentration of ATP in (c) longitudinal and (d) transverse sections is given in a color
scale on the base of the figure (e, embryo; en,
endosperm).
to rise relative to their rates of use as substrates for respiration and biosynthesis (collective data favoring end-products other than starch, e.g. lipids and proteins). This will
include the observation that the starch-type endosperm of
grains appears to be a special modification not broadly observed elsewhere. Other endosperms typically store lipids
(e.g. Arabidopsis, castor bean, etc.) and are peripherally located. However, seed development, oxygen availability, and
the relation between them are likely to vary markedly between species.
Oxygen supply shifts partitioning of labeled sucrose
between different storage products and C-flux to the embryo
Further analysis of oxygen effects on metabolic partitioning
were undertaken using 13C- and 14C-sucrose delivered by a
microliter injection system into the endosperm of intact,
attached kernels. Data from mass spectrometric analyses in
Figure 8(a,b) show that a twofold increase in external O2
resulted in a similar twofold enhancement of 13C-sucrose
conversion to 13C-acetyl-coenzyme A. No significant changes were evident for effects of oxygen enhancement on
transfer of label to 13C-ADP-glucose (Figure 8b), consistent
with metabolite patterns described above. Figure 8(c) shows
that 14C-incorporation into starch was slightly reduced at
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
subambient O2 levels (10%), but was not affected by the
superambient supplies (200%). This line of evidence further
supports above findings on steady-state levels of ADP-glucose, its 13C-labeling, and enzyme activity of ADP-glucose
pyrophosphorylase.
Further analyses were conducted to determine whether
oxygen effects on partitioning between storage products
may have involved an altered capacity for assimilate transfer
to the embryo (the primary site of lipid deposition being the
embryo and its scutellum). Embryos were dissected from
kernels labeled as above, and found to contain nearly
threefold more 14C-assimilates when atmospheric oxygen
levels had been doubled (Figure 8d). Similar increases were
also observed for total label incorporated into embryo lipids
and the pellet (starch, protein, and cell wall) (Figure 8e). On a
whole kernel level, changes due to elevated O2 supply thus
resulted largely from enhanced transfer of label into the
embryo.
Discussion
Oxygen is depleted to hypoxic levels in vivo
Key evidence in this work highlights endogenous oxygen
depletion as a previously unrecognized feature of
76 Hardy Rolletschek et al.
Figure 7. A simplified schematic representation of primary metabolites in
maize kernels (25 DAP) and their response to changing O2 supply. Intermediates of glycolysis and the citrate cycle are shown, as well as branch paths to
related sugars and free amino acids. Arrows indicate probable direction of
predominant C flow. Vertical bars show the level of each metabolite in kernels
after a 6-h treatment with 10, 100 and 200% ambient O2 level (black, dark gray
and gray bars, respectively). Data are given in relative units (mean standard
deviation).
developing maize kernels (Figures 1 and 2). The extent and
duration of this hypoxia are marked. Storing kernels show
strongest O2 gradients immediately inside the peripheral
endosperm, declining to very low levels within the first
200–400 lm. This indicates a high O2 demand that outstrips
the diffusive capacity of the kernel. Hypoxic conditions
Figure 8. Fluxes to metabolic intermediates and storage products in
response to changing O2 supply.
(a, b) Amount of 13C-labeled acetyl-coenzyme A and ADP-glucose, respectively, after delivery of 13C-sucrose (3 ll, 200 mM) into the central endosperm
by an air-sealed minute injection system, and incubation (2 h) of intact kernels
(on ear) at ambient (100%) and superambient (200%) O2 levels.
(c) Partitioning of 14C-sucrose into starch and the remaining pellet (mainly
protein and cell wall) after air-sealed, minute injection of 3 ll 14C-sucrose into
inner endosperm and incubation (6 h) at 10, 100 and 200% of ambient O2 level.
(d, e) Uptake and incorporation of 14C-label into different fractions in maize
embryos as affected by elevated external oxygen. Radiolabeled14C-sucrose
(3 ll) was delivered via air-sealed minute injection into inner endosperm of
kernels attached to the plant. After incubation (6 h) at ambient oxygen levels
(100%) and superambient levels (200%), the embryo was isolated and
analyzed. All data are mean standard error. Significant differences versus
control treatment (100%) are given by *; t-test, P < 0.05).
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 77
Figure 9. Schematic representation of oxygen
and sugar delivery in storing maize kernels.
Oxygen enters the kernel via the entire surface
(blue arrows), and diffuses along steep concentration gradients toward the interior. Oxygen
becomes depleted within the endosperm, but
maintains higher levels within the embryo,
possibly indicating a superior O2 supply. Sugars
(red arrows) enter the kernel via the pedicell/
transfer cell region and flow toward the starchsynthesizing crown area of endosperm and the
lipid-synthesizing embryo via scutellum. High
ATP regions in the endosperm are found adjacent to the embryo base and within starchstoring cells. Within embryo (see insert), high
ATP and sugar supply overlap in regions where
lipid accumulation occurs.
prevail throughout most of the endosperm. Within the embryo, O2 levels are significantly higher. Distinct O2 levels in
embryo and endosperm might be caused by differences in
either O2 supply or respiratory O2 consumption. Lower respiratory activity of the embryo is barely to be expected due
to the requirement of ATP for lipid and protein synthesis. A
superior O2 supply to the embryo seems more likely. In dicot
seeds, the region of seed coat attached to the embryo axis
(root pertrusion zone) is the main site of gas exchange
(Wager, 1974). O2 content in maize endosperm falls to
very low levels (<0.1%), far below those observed in green
seeds of any other species examined thus far (Vicia/Pisum:
Rolletschek et al., 2002a, 2003; oilseed rape: Vigeolas et al.,
2003). Only the chlorophyll-free endosperm of other cereals
approach such low O2 levels (Rolletschek et al., 2004a).
Maize kernels may thus be more susceptible to oxygen
depletion and internal hypoxia than are many developing
seeds and/or have developed specific adaptations.
Maize kernels lack photosynthesis, unlike seeds and
grains of many other crop plants. Oxygen supply to developing kernels thus depends entirely on diffusion from the
ambient air, so is governed by diffusive resistance of tissues,
O2 concentration gradients, and atmospheric O2 level.
Oxygen profiles suggest that O2 uptake occurs via the entire
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
seed surface (see Figure 9). Oxygen flow from pedicell
layers toward endosperm might also be possible because of
elevated O2 levels in these layers. Concentration gradients
further suggest that oxygen flows from the embryo toward
the endosperm, thereby oxygenating the embryo surrounding region. This may have an important regulatory function
for assimilate uptake into the embryo as discussed below.
Oxygen supply via phloem/xylem seems negligible because
both mass flow rates and O2 concentrations within this
liquid (VanDongen et al., 2003) are very low compared with
overall respiratory O2 demand.
ATP gradients reflect metabolic state of tissues, and are
O2-responsive
Data revealed markedly steep ATP-gradients in developing
maize kernels (Figures 3 and 4). In endosperm, this distribution also corresponded to growth and maturation patterns. Cell divisions cease first in central endosperm (Kowles
and Phillips, 1988), followed by endoreduplication of their
DNA (Schweizer et al., 1995), and cell expansion (Larkins
et al., 2001; Arabidopsis; Kondorosi et al., 2000). This process extends from the endosperm crown to the basal
transfer cells (Larkins et al., 2001; Vilhar et al., 2002), and is
78 Hardy Rolletschek et al.
closely associated with cellular differentiation (Nagl et al.,
1985). Starch accumulation occurs as cells expand, whereas
cell division continues only in peripheral cell layers, away
from the embryo (Larkins et al., 2001). Finally, cell death is
initiated in the central and upper endosperm, progressing
toward the base and periphery of the kernel (Young et al.,
1997). Along this developmental gradient, we found that low
ATP was associated with developmentally younger tissues
that maintain some mitotic activity and accumulate starch at
low rates (Figures 3 and 4). ATP levels rose with the extent of
cell differentiation, endoreduplication, and starch accumulation. Two apparent exceptions involved low ATP levels in
mature cells, but the first of these were endopolyploid cells
in the central endosperm that had begun to die, and the
second were cells in basal regions specialized for solute
transport, but not starch accumulation (BELT region with
distinct genetic regulation, Hueros et al., 1999). Overall,
topographical evidence showed that ATP concentration was
apparently coupled to gradients in cell size and endoreduplication (Larkins et al., 2001), expression of ADP-glucose
pyrophosphorylase (Brangeon et al., 1997), starch accumulation (Young et al., 1997), and associated demands for
nucleotide precursors and metabolic activity (Baluka and
Kubica, 1992). A similar relationship was previously identified for tissue gradients in ATP and local onset of starch
storage in the barley caryopsis (Rolletschek et al., 2004a).
Results of both studies support the suggestion that high
local energy levels are indicatory for the metabolic status of
the tissue, and may represent an important feature of
expanding cells undergoing endopolyploidization and
storing starch. The physiologic importance of this observation has to be further investigated.
ATP gradients in the embryo axis and scutellum also
corresponded with those of storage deposition, but primarily for lipids in this instance (Figures 3 and 4). No association
was evident between this ATP distribution analyzed here
and patterns of cell division (Jose-Estanyol et al., 1992;
Stiefel et al., 1999), without endopolyploidization (Killian
et al., 1998). Storage processes in the scutellum begin in the
developmentally older basal regions and proceed toward
the apex, first with starch deposition, and then lipids
(Perdomo and Burris, 1998). A gradient in lipid accumulation
was also evident with the maximum concentration nearest
the endosperm interface (Figure 4f), and this correlated with
ATP distribution. In contrast, oxygen gradients in the
scutellum (Figure 4f) were inverse to those of lipids and
ATP (Figure 4f), consistent with high rates of local consumption. Carbohydrates used for lipid biosynthesis move
within the scutellum from the endosperm interface toward
the interior cells (Griffith et al., 1987; Matthys-Rochon et al.,
1998), and may therefore show gradients similar to those of
lipids and ATP. It is tempting to speculate that zones
favorably supplied with both carbohydrates (from the kernel
interior) and oxygen (from the exterior), could yield the
greatest levels of both ATP and lipids (see Figure 9). In
addition to genetic control of lipid biosynthesis (Boyer and
Hannah, 2001), local energy status may contribute prominently to its in vivo regulation.
Oxygen availability limits energy status and ATP distribution
in maize kernels
A marked influence was demonstrated for normal degrees of
endogenous oxygen depletion, by experimentally altering
oxygen supplies to intact, attached kernels. Data in Figure 5
demonstrate that O2 demand of developing kernels is not
saturated at ambient (atmospheric) O2 levels or even by a
twofold increase in the exogenous supply. Enhanced O2
availability is coupled with higher ATP levels, increased ATP/
ADP ratio, and thus elevated overall energy state of tissues
(Figure 6). Bioluminescence imaging also showed that
increased O2 availability expanded distribution of ATP-rich
zones from the kernel periphery and embryo into the starchstoring endosperm and tissues of the scutellar interior
(Figure 6). A decrease in O2 supply affected the embryo most
strongly (Figure 6c,d), presumably the result of its contrasting metabolic status (sugar composition, storage
metabolism, etc.) relative to endosperm.
Starch biosynthesis in endosperm is adapted to hypoxia, but
lipid synthesis in embryo is limited by in vivo oxygen supply
Further significance of work shown here lies in its demonstration that oxygen depletion inside normally growing
maize kernels is extreme enough to significantly alter pools
of metabolic intermediates (Figure 7) and their partitioning
between storage end-products (Figure 8). Enhancement of
oxygen available to storing kernels increased steady-state
levels of glycolytic intermediates (hexose phosphates, Fruc1,6-diP, PEP, 3-PGA) and citric acid cycle metabolites (acetylcoenzyme A, citrate, isocitrate), as well as some related free
amino acids (Figure 7). Supplies of these precursors and
substrates thus rose relative to their rate of use in biosynthesis and respiration. Conversely, further reductions in
oxygen availability [to 1/10 of normal ambient levels (Figures 6 and 7)], decreased the energy state (ATP/ADP ratio)
and lowered levels of glycolytic intermediates (regardless of
flux effects). Concurrent increases in pyruvate content
would be consistent with further blockage of citrate cycle/
respiration, but did not appear to be accompanied by alanine/lactate fermentation or high ethanolic fermentation.
Some degree of malate formation cannot be ruled out in this
instance (as an NAD-generating end-product of hypoxic
‘glycolysis’), as short distance transfer and subsequent
metabolism could occur without altering overall pool sizes
(Figure 7). More likely, however, is the capacity for endosperm cells to minimize oxygen demand by decreasing
metabolic activity and ATP use during starch storage in a
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 79
manner analog to legume seeds (Rolletschek et al., 2003),
potato tubers (Geigenberger, 2003), and wheat grains
(VanDongen et al., 2004). The lack of metabolic shifts toward
fermentation indicates a well-coordinated dynamic adjustment of kernel metabolism to low O2 availability.
Several lines of evidence indicate that starch biosynthesis
is not oxygen-limited in the normally hypoxic maize endosperm. Increasing the O2 supply did not change either (i) the
steady-state levels of ADP-glucose (Figure 7), (ii) the typically rate-limiting activity of ADP-glucose pyrophosphorylase, (iii) the 13C-labeling of ADP-glucose (Figure 8), or
(iv) the flux of 14C-labeled sucrose into starch (Figure 8).
Furthermore, decreases in oxygen availability to 1/10 of
ambient levels only marginally reduced incorporation of
14
C-sucrose into starch (Figure 8c). Starch biosynthesis may
well represent a key adaptation to the oxygen depletion in
endosperms of developing grains. Points of support include,
first, that the energy demand for starch biosynthesis is
considerably less than that of protein or lipid (Vertregt and
deVries, 1987). Second, starch biosynthesis could contribute
to a potentially critical cycling of PPi and its associated
energy. Under low-oxygen conditions, PPi can maintain
sucrose metabolism via sucrose synthase þ UDP-glucose
pyrophosphophosphorylase (Huber and Akazawa, 1986;
Kavakli et al., 2000). Needed PPi could be generated at sites
of immediate use in cytoplasm during starch biosynthesis,
because of the presence of a uniquely adapted cytosolic
form of ADP-glucose pyrophosphorylase in endosperm of
maize and other cereals (Denyer et al., 1996; Sakulsingharoj
et al., 2004; Salamone et al., 2002). Möhlmann et al. (1997)
further suggested that ATP regulation of starch synthesis
could shift precursor synthesis to the cytosol under conditions such as hypoxia, thus favoring recycling of PPi.
A related adaptation to the low-oxygen environment of
the endosperm environment may be that of sucrose synthase induction by hypoxia (Sachs et al., 1996; Subbaiah and
Sachs, 2001; Zeng et al., 1998). Both SUS1 and SH1 isoforms
are transcriptionally induced by low oxygen in maize [the
SH1 ‘kernel form’ being most responsive to hypoxia (Zeng
et al., 1998)], and O2 levels in the present study altered
enzyme activity and levels of the UDG-glucose product
(Figure 7). Sucrose cleavage by this reversible enzyme is
associated with starch biosynthesis, ATP conservation, and
enhanced sucrose/hexose ratios. Hypoxic induction of
sucrose synthase could thus aid expected metabolite channeling toward starch, shift products of sucrose cleavage
away from hexoses (each requiring an ATP to enter glycolysis), and further favor rising sucrose/hexose ratios (as
result of a high km for sucrose). Still further increases in
sucrose/hexose ratios may result from sucrose resynthesis
in endosperm (Cheng, 1997). Together, these changes could
provide substrates best adapted for respiration, biosynthesis, and compatible sugar signals in an oxygen-depleted
structure (Koch, 1996, 2004; Koch et al., 2000; Zeng et al.,
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
1998). In contrast, oxygen availability clearly limits lipid
biosynthesis. Our tracer experiments showed that elevated
oxygen levels increased conversion of 13C-sucrose to acetylcoenzyme A precursors for lipid biosynthesis (Figure 8a),
and markedly enhanced incorporation of 14C-sucrose into
lipids (Figure 8e). This is consistent with the high energy
demand for lipid biosynthesis itself. Similar data were
shown for seeds of oilseed rape (Vigeolas et al., 2003) where
lipid and starch storage is co-localized in the green embryos.
Collectively, this suggests a general role of oxygen on the
lipid/starch balance in seeds.
Our data also point to a significant role for O2 and ATP in
assimilate transfer to the primary sites of lipid deposition in
embryos (discussed further below). These results, together
with the greater ATP cost for synthesizing lipid and protein
versus starch (Vertregt and deVries, 1987), indicate a
pronounced adaptive advantage for storage of non-starch
reserves in the most well-oxygenated regions at the kernel
periphery (embryo, aleurone, and vitreous layers of endosperm).
Assimilate partitioning to the maize embryo is affected by
local oxygen/energy levels
In general, assimilate partitioning is controlled by gene
expression during the developmental process (Becraft,
2001; Motto et al., 2003). For example, genes coding for
enzymes of glycolysis and fatty acid synthesis in maize
embryos are coordinately upregulated during development
(Lee et al., 2002). Such developmental control is known
to be modulated by the metabolic status of tissues (‘coarse
and fine control’; Koch, 1996; Wobus and Weber, 1999;
Geigenberger, 2003; Scheible et al., 2004).
Significant insights into mechanisms regulating assimilate partitioning toward grain embryos have been difficult
to obtain. Here we demonstrate a causal link between
local O2 availability, ATP levels (supply), and resource
allocation within developing maize kernels. Elevated O2
supply allowing higher respiratory activity enhanced
uptake of 14C-label into the embryo (Figure 8d) and its
subsequent conversion to storage products (Figure 8e).
Energy levels of kernels also increased (Figure 6), and
bioluminescence imaging showed that ATP levels rose in
the embryo-attached region of endosperm and in the
endosperm face of the scutellum (Figure 6c,d), the latter is
a primary route for assimilate transport into the embryo
(Griffith et al., 1987; Matthys-Rochon et al., 1998; see
Figure 9). These data indicate that embryo nutrition may
depend on local ATP levels, particularly at the endosperm/
embryo interface. Although hexose uptake in this context
appears passive, sucrose uptake is energy-dependent
(Griffith et al., 1987) and can progress via sucrose transporters in the maize embryo (Lee et al., 2002). In addition,
even passive entry of hexoses is coupled to energy
80 Hardy Rolletschek et al.
requirements for their initial metabolism by hexokinases.
Activity of these enzymes is typically low in maize kernels,
and associated with lipid accumulation (Doehlert, 1990).
Their activity is critical under oxygen limitation (Bouny
and Saglio, 1996). Another way in which O2 could
stimulate delivery of assimilates to the embryo is by
raising ATP levels that may otherwise limit storage of
proteins and/or lipids in this structure.
In addition to these embryo-localized mechanisms of
oxygen-responsive partitioning, elevated ATP levels within
the embryo-surrounding region might play a role. Sugar
composition is central to embryo nutrition (Griffith et al.,
1987; Matthys-Rochon et al., 1998) and responsive to
enzyme activities in the embryo-surrounding region. Invertases can be active in these sites (Bate et al., 2004), and are
markedly sensitive to O2 (Zeng et al., 1999). Oxygen availability could thus affect delivery of the hexoses preferentially
taken up by the embryo. The link between O2 supply, local
ATP levels and 14C-uptake let us hypothesize that the energy
state at the endosperm/embryo interface may exert a control
function for partitioning of photoassimilates toward the
embryo and, thus, for embryo growth.
Two important insights arise from the novel combination
of approaches used in this study. (These tools allowed a
literally ‘new view’ of gradients across developing endosperm, their interrelationships, and the extent of their
oxygen-limitation in vivo.) First, we show that oxygen
depletion in cells of the interior of growing maize kernels
is far more pervasive than previously recognized. Second,
manipulation of the oxygen supply in the kernel interior
markedly altered (i) energy status, (ii) ATP gradients, (iii)
metabolite levels, (iv) assimilate partitioning to embryos,
and (v) deposition of non-starch storage products.
The significance of these findings is twofold. Although
possibly coincidental, a distinct functional and evolutionary
advantage can be envisioned for sites of processes most
limited by internal oxygen depletion (lipid and protein
deposition) to be located in peripheral tissues of the kernel
(e.g. the embryo and vitreous outer layers of the maize
endosperm). Second, data here and elsewhere suggest that
the starch-type endosperm of maize and other grains is
particularly well-adapted for deposition of storage reserves
under localized oxygen depletion.
Experimental procedures
Plant growth
Maize plants (Zea mays L.) were cultivated in a greenhouse in spring
and summer under natural light supplemented with lamps to provide a 16/8-h photoperiod and an approximate light intensity of
800 lmol photons m)2 sec)1. Temperature was controlled between
28 and 32C. Kernels were harvested at distinct developmental
stages (DAP) during the mid-light phase, snap-frozen in liquid N2
and stored at )80C until analyzed.
Measurement of internal O2 concentration
The O2 concentration inside kernels was measured using oxygensensitive microsensors in a procedure modified from that developed earlier for legume cotyledons by Rolletschek et al. (2002a). For
maize, the intact ear was carefully moved into a fixed position and
anchored firmly for subsequent micromanipulation. To allow entry
of the microsensor to the seed, a small cut was made into the husks.
Oxygen concentration below the husks was near atmospheric levels
independent of light supply as was tested before. The microsensor
was inserted into the intact kernel (remaining on the ear) using a
micromanipulator. The site of sensor entry was sealed with silicone
to prevent oxygen movement along microchannels. Measurements
were carried out within 5 min. The O2 concentration is expressed in
% of atmospheric saturation (21 kPa ¼ 100%). After measurement,
the seed was dissected at the measured transect to identify the exact
position of the sensor.
Imaging of local ATP concentration
Bioluminescence imaging was used to measure ATP distribution
in cryosections of kernels. This technique allows the quantitative, histographical mapping of ATP, and was described in detail
earlier (Borisjuk et al., 2003). Briefly, cryosections were prepared
from snap-frozen kernels, and immersed into an enzyme solution
linking ATP to the reaction of firefly luciferase. The enzyme emits
photons with an intensity proportional to the content of ATP.
The light emission was registered with a photon-counting system
(Hamamatsu, Herrsching, Germany) linked to a microscope. ATP
concentrations determined by bioluminescence are directly proportional to those determined by conventional HPLC [correlation
coefficient 0.85; P < 0.01 (Borisjuk et al., 2003)].
Metabolite analysis
Frozen plant material was rapidly weighed, immediately homogenized with an ice-cold mortar and pestle in liquid N2, and
extracted with trichloroacetic acid as in Rolletschek et al. (2002a).
Starch was determined in the remaining pellet as in Borisjuk et al.
(2002). Aliquots of snap-frozen extracts were used for subsequent
metabolite analyses. Free amino acids were measured by HPLC
(Rolletschek et al., 2002b). Dissolved sugars were measured photometrically (Borisjuk et al., 2002). Adenine nucleotides were
measured after derivatization by HPLC following the procedure of
Haink and Deussen (2003) modified as follows: 20 ll of extract were
incubated with 100 ll chloroacetaldehyde, and 880 ll buffer (62 mM
citrate, 76 mM KH2PO4, pH 5.5) at 80C for 40 min. Derivatized
samples were immediately cooled to 4C until analysis. Chromatographic separation within 4 min and fluorescence detection was
carried out as described by Haink and Deussen (2003). Metabolites
of glycolysis and the citrate cycle were measured by liquid chromatography coupled to mass spectrometry (LC-MS). Chromatography was conducted using a DX-500 ion chromatography system
(Dionex, Sunnyvale, CA, USA). Separation was carried out on a
Dionex AS11-HC column (2 · 250 mm) and an additional guard
column (AG11-HC) at 30C. A binary gradient at a constant flow rate
of 0.5 ml min)1 was applied using 100 mM sodium hydroxide (eluent B) and distilled water (eluent A). The gradient was produced by
linear concentration changes: these were initiated with 20% B,
raised to 34% B during the first 7 min, 70% B in the next 7 min, and
to 100% B in the final 1 min. After holding at 100% B for 4 min, levels
were returned to 20% B over a 2-min period and equilibrated for
10 min. Column effluents were directed to an ASRS-ULTRA (2-mm)
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
Positional cues for resource partitioning 81
anion self-regenerating suppressor (Dionex) working in the external
water mode (2 ml min)1) at 100 mA. After passing the conductivity
cell, the effluent was split (1:1), and directed into the electrospray
chamber. MS analysis was performed using a triple quadrupole
(LC1200; Varian, Palo Alto, CA, USA) using the following parameters: ESI N2 pressure 51 psi, needle )5000 V, shield )200 V, drying
gas 225C and 18 psi, detector voltage 1500 V, mass peak with
0.7 amu, scan time 2.5, and detection in the negative ion mode.
Metabolites were measured in parallel using single-ion monitoring
mode scanning the deprotonated ions [M–H]) except for the following (scanned masses in parenthesis): acetyl-coenzyme A (264.9),
ADP-glucose (293.6), UDP-glucose (282), citrate/isocitrate (173),
malate (115), phosphoenolpyruvate (166.7), and fructose-1,6-diphosphate (168.8). The validity of the method was checked using
known standards (Sigma, St Louis, MO, USA) and by previous
recovery experiments with different tissues/species (Rolletschek
et al., 2004a,b). Relative quantification was carried out using scanned ion traces. Data were normalized to plant mg fresh weight and
to an internal reference (13C-labeled pyruvate; Isotec, Miamisburg,
OH, USA) added during the extraction of each sample.
Determination of enzyme activities
ADP-glucose pyrophosphorylase (AGPase; E.C. 2.7.7.27) and
sucrose synthase (E.C. 2.4.1.13) activities were assayed using coupled spectrophotometric assays as described earlier (Rolletschek
et al., 2002b). Enzyme assays were tested for substrate dependence
and substrate and linearity with time and amount of extract.
Histological methods
Iodine staining was used to visualize starch granules (Borisjuk et al.,
2002). Lipid staining was conducted using fresh, free-hand sections
with Sudan B as described by Brundrett et al. (1991). Briefly, Sudan
red B (Sigma) was dissolved in polyethylene glycol (400D; Sigma)
by heating at 90C for 1 h, and an equal volume of 90% (v/v) glycerol
was added. Sections were stained for 24 h at room temperature,
then rinsed with deionized water, fixed in 70% ethanol, and photographed.
Acknowledgements
Oxygen treatment
To study the effect of altered oxygen supply, kernels on an intact ear
(enclosed within a transparent plastic chamber) were aerated with
gas mixtures (combined by a multigas controller, Type 647B; MKS,
Muenchen, Germany) containing atmospheric oxygen levels (about
21 vol.%, referred to as to 100%), 42 vol.% O2 (¼200%) and 2.1 vol.%
O2 (¼10%, balanced by N2), respectively, at a flow rate of 2.5 l min)1.
After a 6-h incubation, kernels were quickly removed, immediately
frozen in liquid N2, and stored at )80C until used for analysis. The
multigas controller was also used for stepwise increases in the
external oxygen supply (see Figure 5).
We are grateful to U. Tiemann, K. Lipfert and K. Blaschek for
excellent technical assistance, C. Klukas and F. Schreiber for mapping metabolic networks as well as M. Hajirezaei for help with
14
C-isotope studies.
Supplementary Material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/TPJ/TPJ2352/
TPJ2352sm.htm
Table S1. Primary metabolites and their response to changing O2
supply. Data are given as mean standard deviation.
Labeling experiments
For monitoring of in vivo fluxes, developing kernels on an intact ear
were labeled with either stable (1-13CFru-sucrose; Omicron Biochemicals, South Benol, IN, USA) or radioactive (U-14C-sucrose;
Amersham-Buchler, Freiburg, Germany) tracers, and treated with
different gas mixtures as described above. Using a microsyringe, 3 ll
of labeled 14C-sucrose (7.4 MBq ml)1) were injected into the endospermal conducting tissue of developing kernels (25 DAP) on intact
ears. The site of needle entry was sealed with silicone to prevent gas
exchange. After a 6-h incubation time during the light period, the
kernels were removed and immediately frozen in liquid N2. Subsequently, an extraction procedure was carried out yielding a water/
ethanol-soluble fraction, starch fraction and the remaining pellet as
described earlier (Rolletschek et al., 2002b). A modified method was
used for the determination of label uptake by the embryo and partitioning into lipids. The kernels (30 DAP) were labeled and treated as
above. After a 6-h incubation, the embryo was rapidly removed from
the kernel, rinsed quickly in distilled water, and immediately frozen in
liquid N2. After weighing, the kernels were extracted as in Bligh and
Dyer (1959) giving a chloroform-soluble fraction (lipids), water/
methanol-soluble fraction and the remaining pellet. For determination of the labeling pattern within metabolic intermediates, 3 ll of
labeled 13C-sucrose (200 mM) were injected into the endospermal
conducting tissue of intact kernels (25 DAP) as above. After a 2-h
incubation, the kernels were removed quickly, immediately frozen in
liquid N2, and subsequently extracted using trichloroacetic acid as
described above. Amounts of 13C-labeled acetyl-coenzyme A and
ADP-glucose were quantified by mass spectrometry.
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 69–83
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