Review
Blackwell
Oxford,
New
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©
1469-8137
0028-646X
February
10.1111/j.1469-8137.2008.02752.x
2752
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0
Tansley
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ThePhytologist
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The oxygen status of the developing
seed
Author for correspondence:
Hardy Rolletschek
Tel: +49 39482 5686
Fax: +49 39482 5500
Email: [email protected]
Ljudmilla Borisjuk and Hardy Rolletschek
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D–06466
Gatersleben, Germany
Received: 1 October 2008
Accepted: 3 December 2008
Contents
Summary
17
VI.
Gene expression and metabolism under low oxygen
24
I.
Introduction
17
VII. Mechanisms of oxygen sensing and balancing in seeds
25
II.
Oxygen diffusion and the barriers to gas exchange
18
III.
Metabolic indicators of seed hypoxia
19
VIII. Might low internal oxygen levels be advantageous
for seed development?
27
IV.
High-resolution mapping of oxygen distribution in the
developing seed
20
The effect of environmental factors on steady-state
oxygen concentrations
23
V.
Acknowledgements
27
References
28
Summary
New Phytologist (2009) 182: 17–30
doi: 10.1111/j.1469-8137.2008.02752.x
Key words: hypoxia, nitric oxide (NO), oxygen
diffusion, oxygen sensing, seed development,
seed photosynthesis, storage metabolism.
Recent applications of oxygen-sensitive microsensors have demonstrated steep
oxygen gradients in developing seeds of various crops. Here, we present an overview
on oxygen distribution, major determinants of the oxygen status in the developing
seed and implications for seed physiology. The steady-state oxygen concentration
in different seed tissues depends on developmental parameters, and is determined to
a large extent by environmental factors. Photosynthetic activity of the seed significantly
diminishes hypoxic constraints, and can even cause transient, local hyperoxia.
Changes in oxygen availability cause rapid adjustments in mitochondrial respiration
and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen
balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular
approaches aiming to increase oxygen availability within the seed are discussed.
Abbreviations: At, Arabidopsis thaliana; AOX, alternative oxidase; COX, cytochrome C oxidase; HIF1, hypoxia-inducible factor; NO, nitric oxide; ROS, reactive
oxygen species.
I. Introduction
The modern atmosphere contains approx. 21 kPa oxygen.
However, over the course of the past 550 million yr (Phanerozoic
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time), during which time the vascular plants invaded the land
surface, plants have adapted to levels of atmospheric oxygen
ranging from 13 to 51 kPa (Raven, 1991). This variation has
been a major driver of plant evolution, and has led to the tuning
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of plant architecture/ultrastructure and metabolism to tolerate
both low and high oxygen supply (Berner, 1999). Although
over a shorter time-scale the atmospheric oxygen level may
appear stable, plants must be able to adapt to variation in
oxygen provision imposed by the local environment. For
example, little oxygen is available to the plant root in a temporarily
waterlogged soil, so plants have developed a number of strategies
for acclimatization, avoidance and escape (Armstrong et al.,
1994; Crawford & Brändle, 1996; Drew, 1997; Vartapetian
et al., 2008). The diffusion of gas is 10 000 fold slower through
a liquid medium than through air, so waterlogging rapidly
leads to hypoxic and eventually even to anoxic conditions. Under
hypoxia, the concentration of oxygen limits mitochondrial ATP
production (oxidative phosphorylation), whereas under anoxia
there is essentially no oxygen available for mitochondrial respiration. A restricted capacity for oxygen diffusion, in conjunction
with a high rate of cellular metabolism, can generate hypoxia
even in aerial organs such as the fruit (Ke et al., 1995), certain
vascular tissues (Kimmers & Stringer, 1988), the pollen grain
(Leprince & Hoekstra, 1998) and the seed (Rolletschek et al.,
2002).
From a historical perspective, seed germination and subsequent seedling growth has been one of the classical issues of
hypoxia-related research (Al-Ani et al., 1985; Corbineau &
Côme, 1995). This review focuses on developing seeds where
the establishment of hypoxic conditions can affect the developmental pattern of gene expression, in vivo enzymatic activity,
metabolite pool sizes and metabolic fluxes. The significance of
studies on hypoxia in developing seeds is based on the role of
seeds in human and animal nutrition. Assuming that endogenous hypoxia causes restrictions in overall metabolic activity
and eventually seed yield, there are considerations to lessen and/
or to prevent hypoxic constraints. However, such approaches
require detailed knowledge of the factors determining the
hypoxic strength, the topography (which seed tissue is actually
hypoxic) and the possible advantages of low internal oxygen
levels. Of course, there is always the possibility that changes
in the development of the seed can have consequences for
later stages of the life cycle (i.e. post harvest viability and
germination). Recent studies on seed photosynthesis have
indicated a role for alleviating and/or preventing hypoxia
inside the seed (Rolletschek et al., 2005a). Most but not all
seeds become green during development, and this raises
the questions as to why not all seeds have photosynthetic
abilities. Are there environmental conditions that favour
nongreen seed tissues? Are the evolutionary costs too high for
some species?
This review describes the significance and possible regulatory
functions of hypoxia for seed growth and physiology. It evaluates the normal occurrence of oxygen limitation in developing
seeds, compares the oxygen distribution (termed ‘oxygen maps’)
in a range of crop seeds and, finally, discusses the mechanisms
for oxygen balancing and their implications on the determination of seed growth.
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II. Oxygen diffusion and the barriers to gas
exchange
Certain structural features of the developing seed affect their
gas exchange capability. The outermost layers of seed (the cuticle,
the dense epidermal or compressed cells of the pericarp and
the seed coat) effectively form a sealed space separated from
the external environment. The outer epidermal layers of the
dicotyledonous seed coat, for example, have no functional
stomata (Geisler & Sack, 2002). In the monocotyledonous seed,
a low stomatal frequency is the norm (Cochrane & Duffus,
1979). Such a surface is a barrier to gas exchange (Nutbeam
& Duffus, 1978; Sinclair et al., 1987, Sinclair, 1988) and acts
to restrict diffusion to the micropylar, funicular or vascular region
(Wager, 1974a; Sinclair, 1988). Distinct lipid-containing or
suberized cell layers, sometimes termed ‘barriers’, are commonplace inside the seed (Freeman & Palmer, 1984). The term
‘barrier’ is frequently associated with semipermeability (Cochran,
1983; Welbaum & Bradford, 1990; Beresniewicz et al., 1995),
although neither their features nor their role in the context of gas
exchange are well understood. Cuticles differ in their permeability
for particular gases, with carbon dioxide diffusing much more
readily than oxygen. Diffusive transport of CO2 may occur to a
large extent via the liquid phase as the solubility of CO2 in water
is much higher than that of O2 (Ho et al., 2007). Permeability
probably also varies from organ to organ, from species to species,
and during development (Lendzian & Kerstiens, 1991).
The biochemical composition of ‘semipermeable’ membranes
may determine their resistance to gas exchange. The perisperm
layer (‘the membrane envelope’ of the embryo) present in
many seeds is a characteristic example. It consists of a number
of callose-containing layers deposited outside the outer walls
of the endosperm cells, and acts as a molecular sieve (Yim &
Bradford, 1998; Ramakrishna & Amritphale, 2005). However,
the lipid-containing layer of the envelope (a chloroform-soluble
waxy layer, stainable with Sudan dye) is not required for semipermeability, so its role is unclear. The barrier function of certain
structures within the sunflower (Helianthus annuus) seed has
been analysed recently. The step-by-step peeling of the peripheral tissue layers was combined with the monitoring of localized internal oxygen level (Fig. 1; Rolletschek et al., 2007). The
surprising conclusion was that the major barrier to diffusive
oxygen uptake was the very thin lipid-rich membrane covering
the oil-storing embryo, while the much thicker multiple layers
of the pericarp and attached seed coat were ineffective in controlling gas exchange. This finding emphasizes the importance
of chemical composition as opposed to tissue thickness as the
major determinant of oxygen diffusibility. Thus, even the very
small seeds of the model plant Arabidopsis thaliana (At) may
be prone to hypoxia, with a small reduction in external oxygen
being sufficient to induce hypoxic responses (Gibon et al., 2002).
Molina et al. (2008) analysed the chemical composition of the
cuticular layers of both At and rapeseed (Brassica napus) seed,
and found that the suberized layers are associated with the
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Fig. 1 Structure of sunflower seeds and its
effects on internal oxygen concentration.
(a) Longitudinal section through seed. (b)
Fragment of outermost tissues of seed:
multiple cell layers of pericarp, attached seed
coat and membrane envelope (arrows)
covering the embryo. Toluidine blue staining
of fragment from (a). (c) Visualization of
lipidous compounds by staining with Sudan
dye (shown in red). Insert shows changes in
the internal oxygen level in embryo of intact
seeds, and after removal of pericarp and
membrane envelope. em, Embryo; pe,
pericarp; sc, seed coat.
outer integument, while layers of cutin-like polyesters are found
attached to the inner one. The deposition of both is initiated
at mid-maturation and is not associated with the earlier deposition of waxes. Substantially lower levels of polyester monomer
are present in the chalazal region (including the scar of funiculus
and micropyle) than in the nonchalazal region of the seed coat,
which may result in less diffusional impedance of oxygen in
this region. This would correspond to the situation in pea
(Pisum sativum), where it has been known for many years that
gas exchange through the seed coat occurs largely through the
micropylar region (Wager, 1974a).
Seeds lack an enhanced gas-space distribution system, such
as the aerenchyma present in some vegetative tissues (Justin &
Armstrong, 1987). Thus, their gas exchange capability depends
mainly on a combination of the structural arrangement of cells
and intercellular spaces, and on the existence of distinct gas
diffusion barriers. A detailed study of the three-dimensional
structure of mature A. thaliana seed cells, and especially the
intercellular air spaces, has been achieved using synchrotron
X-ray tomography (Cloetens et al., 2006). Air channels, which
traverse both the hypocotyl and cotyledons, have been identified
in developing seeds (B. Nicolai, pers. com.); and these channels
are interconnected, as they are also in dry A. thaliana seeds
(Cloetens et al., 2006) and certain fruits (Verboven et al., 2008).
A high level of interconnectivity between these air spaces clearly
serves to increase tissue porosity, thereby facilitating gas exchange
within the developing/germinating embryo. The genetic basis
of this network remains to be elucidated, as does the question
© The Authors (2009)
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as to whether the environment plays any determining role. In
the root, the conformation of the intercellular air space can be
influenced by a number of environmental stresses, with implications for gas exchange and respiration (van Heerden et al., 2008).
III. Metabolic indicators of seed hypoxia
There has long been a body of albeit indirect evidence for the
existence of oxygen-depleted zones within the developing seed.
For example, Boyle & Yeung (1983) noted the activity of alcohol
dehydrogenases in bean (Phaseolus vulgaris) seeds, while Wager
(1974b) observed the release of ethanol from pea seeds, and
Gambhir et al. (1997) observed a decline in the cytosolic pH
in the seeds of both soybean (Glycine max) and mustard (Brassica
juncea) during certain stages of seed development. When soybean
seeds were provided with oxygen, there was a shift in respiratory
activity, along with a characteristic change in the ratio of adenine
nucleotides (Gale, 1974; Shelp et al., 1995). The observation
that both the number and unit size of the seed depend on the
atmospheric oxygen level (A. thaliana, Kuang et al., 1998; rice
(Oryza sativa), Akita & Tanaka, 1973; soybean, Quebedeaux &
Hardy, 1975; wheat (Triticum aestivum), Musgrave & Strain,
1988) underlines the importance of hypoxia. Substantial oxygen
concentrations in the atmosphere seem necessary to drive the
diffusion of oxygen into the seed. Failing the establishment of
these, the influx is too low to meet the demand, leading to
abnormal seed development and even seed abortion. Attempts
have been made to model oxygen availability to the plant embryo
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(Collis-George & Melville, 1974; Dungey & Pinfield, 1980),
but technical difficulties in obtaining an accurate measurement
of localized concentrations of oxygen within the seed have
hampered the development of predictive respiration and oxygen
diffusion models.
IV. High-resolution mapping of oxygen
distribution in the developing seed
Porterfield et al. (1999) used miniature glass electrodes to
measure the internal oxygen concentration in the siliques of
At and rapeseed, and reported minima of 6.1 kPa and 12.2 kPa,
respectively. Although these values were still relatively high,
hypoxia was, nevertheless, suggested to be an important factor
for seed development. Direct estimates of seed oxygen concentrations has only become possible of late, and have been made
for a range of crop species, including barley (Hordeum vulgare),
broad bean (Vicia faba), maize (Zea mays), pea, rapeseed,
soybean, sunflower and wheat. The use of oxygen-sensitive
microsensors with a tip diameter of 50 µm has allowed for a
high degree of spatial resolution of steady-state oxygen levels.
Such microsensors have been used to generate oxygen maps, to
detect dynamic changes in tissue oxygen levels in response to
environmental factors, and to measure the rate of release of
photosynthetic oxygen. In the following, we review the outcomes
of applying microsensors to seeds (but leave the reader to refer
to the cited papers to obtain descriptions of the methodology).
1. Nongreen seeds
Seeds of sunflower and maize remain nongreen throughout their
development, and thus they do not produce endogenous photosynthetic oxygen. This has some implications for the oxygen status
of the seed. The main storage organ of sunflower is the embryo.
A representative oxygen concentration profile is shown for a
sunflower seed at the main storage stage (Fig. 2a; Rolletschek
et al., 2007). Within the first 500 µm of the micropylar region,
the oxygen level declines to approximately half-saturation values,
and falls dramatically within the lipid-storing embryo, where
strongly hypoxic levels of below 1 µm prevail, i.e. approximately
0.08 kPa partial pressure. (Please note that c. 258 µm corresponds
to atmospheric saturation (100%) at 25°C. However, this concentration value is probably an overestimate because cell sap
contains significant amounts of soluble compounds, which in turn
reduce oxygen solubility). The oxygen level of the seed tissue was
not responsive to light, as expected, given that no chlorophyll
is present in the seed. The mean oxygen concentration displayed
a clear developmental pattern, with the lowest levels occurring
between 10 d and 25 d after flowering (Rolletschek et al.,
2007). This timing corresponds to the periods of maximal oil
accumulation rate (Luthra et al., 1991). Sunflower seeds accumulate large amounts of oil within the embryo. The implications
for fatty acid composition of the oxygen status in the seed are
described in more detail by Rolletschek et al. (2007).
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Fig. 2 Representative oxygen maps for developing seeds of
(a) sunflower, (b) maize and (c) barley. Oxygen was measured using
microsensors along the x-axis (penetration depth given in µm). The
O2 concentration is given in µM (258 µM corresponds to atmospheric
saturation at 25°C). The O2 profiles were measured in either light
(red circles) or dark (black circles). Chlorenchym is indicated by green
colour; em, embryo; en, endosperm; pe, pericarp.
In maize caryopses, the starchy endosperm is the major
storage organ, but the embryo also accumulates both lipid and
protein (Doehlert, 1990). The environment of the developing
caryopsis is clearly hypoxic, with the internal oxygen level
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dropping dramatically within the first 500 µm of the surface
(pericarp/aleurone) (Fig. 2b; Rolletschek et al., 2005b). The
remainder of the endosperm contains little detectable oxygen,
with mean levels in the interior of c. 4 µm. The oxygen level
increases at the endosperm–embryo interface (the innermost
scutellar surface), reaching approx. 50 µm at the outer surface
of the embryo. This gradient suggests diffusion of oxygen from
the embryo towards the oil-storing scutellum and the embryosurrounding region, and could be associated with a regulatory
function for assimilate partitioning and local storage activity
within the seed (see Section VI). In maize, processes most limited
by internal oxygen depletion (lipid and protein storage) are
located in peripheral tissues of the seed (Rolletschek et al.,
2005b). Although possibly coincidental, this topography provides functional and evolutionary advantages.
Nongreen seeds do not have the capacity for endogenous
oxygen production, and thus rely entirely on diffusive oxygen
uptake. However, a residual level of oxygen remains detectable,
indicating a regulatory mechanism for the avoidance of anoxia
in the seed (see Section VII).
2. Seeds having a green pericarp
The endosperm of the wheat and barley caryopsis accumulates
large amounts of starch, while assimilate storage in the embryo
is negligible. Chlorophyll is present in a distinct layer within
the pericarp (the so-called chlorenchyma), which covers the
whole endosperm, except for the region of the vein and the
nucellar projection (Rolletschek et al., 2004). Photosynthesis
is initiated in barley at 4–8 d after fertilization, corresponding
to an intermediate growth phase (Wobus et al., 2004). Detailed
oxygen maps are available for the barley caryopsis (Rolletschek
et al., 2004), of which a representative, taken from a caryopsis
10 d after fertilization (early storage phase), is shown as Fig. 2c.
When measured in the dark, the oxygen concentration fell only
slightly across the pericarp but dropped dramatically towards
the interior region where the nucellar projection, endospermal
cavity and endospermal transfer cells are located. These tissues
represent an essential part of the main transport route for
assimilates to the starchy endosperm (Patrick & Offler, 2001).
When measured in the light, the reduction in oxygen level was
less drastic. The oxygenation of transport-related tissues via seed
photosynthesis is assumed to be important for the control of
assimilate uptake (see Section VI). In the later developmental
stages, the lowest oxygen concentrations were present in the
central endosperm (< 0.3 µm and 3 µm under unlit and lit
conditions, respectively). Between the pre-storage and major
storage stages, the mean endospermal oxygen level declined,
which was reflected in a decreased cell energy status (Rolletschek
et al., 2004) and the induction of fermentation enzymes
(Macnicol & Jacobsen, 2001). During the storage phase where
photosynthetic activity is at its peak, the oxygen level within
the chlorophyll strands of the pericarp can reach up to 600 µm
(hyperoxia), reflecting a high rate of local oxygen evolution.
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The form of the concentration gradients suggests that the oxygen
produced diffuses both outwards towards the seed surface as
well as inwards towards the centre of the endosperm. This
finding is consistent with the well-known phenomenon of
oxygen release from illuminated caryopses (Nutbeam & Duffus,
1978).
Less detailed information is available for the wheat caryopsis.
A gradient of oxygen concentration (from the outside to the
inside) was observed in caryopses 20 d after fertilization harvested during the light period, with a level at 2 mm from the
surface of approx. 25 µm (van Dongen et al., 2004). Given the
overall similar seed structure and physiology of wheat and
barley, it may be expected that oxygen maps would also be
rather similar to one another.
3. Seeds having a green embryo
In legume seeds, the embryo becomes fully green during its
early development. The broad bean, pea and soybean share a
characteristic oxygen distribution in their seeds: oxygen concentrations decline across the seed coat and reach minimum levels
within the endospermal liquid. In the embryo proper, oxygen
level is low in darkness, but increases under light conditions
(Rolletschek et al., 2002, 2003, 2005a). A characteristic oxygen
concentration profile measured in a developing seed of broad
bean under light is shown in Fig. 3a. The oxygen level declines
dramatically beyond the outer layer of the seed coat, suggesting
that oxygen entry from the surrounding air space into the seed
is restricted. The lowest oxygen levels (as little as 3 µm) occurred
within the endosperm vacuole, between the seed coat and the
embryo. Within the embryo, the oxygen concentrations rose to
higher values, indicating endogenous oxygen release via photosynthesis (Fig. 3a) but remained low under darkness. Thus the
oxygen status of the embryo is clearly influenced by the light
supply. In the presence of light, the increase in oxygen level was
reduced at the earlier stages of development, as the photosynthetic activity of the embryo increases with its age. The peak
rate of photosynthetic oxygen release was 30–60% of the seed’s
overall oxygen demand. When measured in the dark, the mean
oxygen level within the embryo was c. 9 µm, with no pronounced
gradients in oxygen concentration.
The pattern of oxygen distribution in the pea seed
(Rolletschek et al., 2003) was similar to that in the broad
bean, with a strong decline across the seed coat towards the
interior, the lowest levels within the endosperm cavity and
cotyledons (2–8 µm), but with a considerable increase upon
illumination (up to 130 µm).
Soybean seeds are more sensitive to lighting conditions as
compared to pea/broad bean. When seeds are illuminated, the
oxygen level within the embryo rises from c. 2 µm (in dark) up
to 220 µm (under full light, Fig. 3b; Rolletschek et al., 2005a).
Oxygen distribution shows a gradient towards the centre of
the tissue. This pattern is consistent with the observation that
photosynthetic capabilities are greater in the outer than in the
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inner regions of the cotyledon (Borisjuk et al., 2005). Gradients
in photosynthetic activity across the seed are based on higher
light supply to outer versus inner regions. In addition, there are
pronounced gradients in photosynthetic abilities as shown by
pulse-amplitude-modulated fluorescence analysis. An image
of the effective quantum yield of photosystem II is shown in
Fig. 3c (Borisjuk et al., 2005), indicating a clear gradient in
photosynthetic abilities towards the centre. This pattern is
based on ultrastructural characteristics of plastids and results
in distinct photosynthetic oxygen release rates in inner versus
outer regions of the soybean seed.
When seeds adapt to illumination, the internal oxygen level
can fall back to the steady-state nonilluminated level. This
abrupt decrease in oxygen level probably reflects a concomitant
rise in the respiratory oxygen demand. Soybean embryos at a
later developmental stage display less dramatic variation in
internal oxygen concentration, and a reduced compensatory
increase in respiration. Thus, the capacity to balance oxygen
consumption with its supply declines with maturation. This
pattern is characteristic for soybean embryos, but is much less
pronounced in pea or broad bean.
Rapeseed is grown as a source of oil, but its seed also accumulates considerable amounts of protein. Vigeolas et al. (2003)
analysed rapeseed and found internal concentrations of approx.
10 µm. However, there were no indications on the positioning
of the sensor within the seed and the amount of light. Detailed
oxygen maps have been generated in our own group (unpublished). Figure 3c shows a representative profile for a seed at
main storage stage (for descriptions of the methodology,
please see Rolletschek et al., 2002). In the dark, the oxygen
concentration inside the seed declined gradually, reaching a
minimum of approx. 1 µm within the inner cotyledon and the
endosperm liquid in the centre of the seed. The seed coat and
outer cotyledon remained well oxygenated (> 75 µm). The
seed has a much greater photosynthetic capacity than either
legume or cereal seed, with the result that there are strong
changes in oxygen levels during the light–dark switch (see
Section V).
In summary, the oxygen level in the growing green embryo
is controlled by its developmental stage and affected by the
external lighting conditions experienced by the plant. The
sensitivity to these factors is species-dependent.
Fig. 3 Representative oxygen maps and photosynthetic parameters
of green seeds. (a) oxygen map of broad bean seeds measured under
light; (b) oxygen map of soybean seeds measured under light (red
circles) or dark (black circles) conditions; (c) image of the effective
quantum yield of photosystem II (Φ PSII) measured in soybean seeds
at 63 µmol quanta m−2 s−1, yield is indicated by the colour bar;
(d) oxygen map of rapeseed measured in darkness. Oxygen profiles
were measured using microsensors along the x-axis; O2
concentration is given in µM. em, Embryo; ev, endospermal vacuole;
ic, inner cotyledon; oc, outer cotyledon; ra, radical; sc, seed coat.
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V. The effect of environmental factors on
steady-state oxygen concentrations
In most dicotyledonous species, including A. thaliana, the main
storage organ (the embryo) becomes green and photosynthetically active during early seed development. The embryo is
surrounded by both the pod/silique and the seed coat. The
atmosphere inside the pod/silique provides an environment
characterized by an elevated carbon dioxide and lowered oxygen
concentration compared with the external atmosphere, thanks
to carbon dioxide release and oxygen uptake by the metabolically
active seed (Shinano et al., 1995). Depending on light supply,
the ‘seed environment’ changes, as the carbon dioxide level falls
and the oxygen level increases (Porterfield et al., 1999). In soybean, approx. 10% of incident light reaches the embryo surface
(Rolletschek et al., 2005a), and a similar proportion applies for
white lupin (Atkins & Flinn, 1978) and rapeseed (Eastmond
et al., 1996). Although light availability for the green embryo
is rather limited, photosynthetic activity within the seed generates
rapid changes in internal oxygen levels (Fig. 4a, Rolletschek et al.,
2002). In soybean, stepwise increases in light supply resulted
in a several-fold increase in the photosynthetic oxygen release
rate and concomitantly increased the steady-state oxygen concentration within the embryo (Fig. 4b; Rolletschek et al., 2005a).
The most dramatic changes observed in the seed oxygen status
to date involve rapeseed (Fig. 4c; Borisjuk et al., 2007), where
the light–dark switch generates large, reversible and rapid fluctuations in internal oxygen levels, ranging from strong hypoxia
(< 1 µm) to severe hyperoxia (> 700 µm). After light adaptation,
the oxygen concentration in illuminated seeds can fall significantly, as described for soybean (see above).
Despite the low level of light available inside the seed, the
level of photosynthetic activity, as measured by oxygen release,
is surprisingly high. This is thought to reflect the specialized
nature of chloroplasts present in the seed, whose thylakoids
contain chlorophyll–protein complexes similar to leaf chloroplasts, but exhibit a greater proportion of granal stacking (Saito
et al., 1989; Asokanthan et al., 1997). This adaptation can
generate distinct light harvesting properties and low saturation
levels for photosynthetic electron transport (Borisjuk et al.,
2005). The ratio of Rubisco to total chlorophyll is low in the
seed (Ruuska et al., 2004) and is similar to that present in shadeloving leaves. It has been hypothesized that the plastids have
become specialized to use light reactions to generate the ATP/
NADPH required to fuel biosynthesis of the various storage
products. The embryo plastids have been classified as photoheterotrophic organelles (Asokanthan et al., 1997), which are
structurally and metabolically adapted to drive photosynthetic
reactions at low light intensities. Thus their activity affects the
oxygen status of the seed, determines its energy and redox state
and eventually its biosynthetic flux (Browse & Slack, 1985;
Ruuska et al., 2004; Rolletschek & Borisjuk, 2005).
Seed photosynthesis can deliver significant amounts of oxygen, alleviating potential hypoxic conditions. Thus, green seeds
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Fig. 4 Dynamics of oxygen status in response to light supply. (a) The
O2 concentration measured in pea cotyledons in light interrupted by
two dark intervals. Arrows indicate the start of the 30 s dark period.
(b) The O2 concentration measured in soybean cotyledons (circles,
left axis) and photosynthetic O2 release rate (bars, right axis; data are
given in nmol g−1 FW min−1) in response to light intensity. (c) Timecourse of O2 concentration in rapeseed measured during light–dark
transitions.
have a means to stimulate their own metabolic activity during
the light phase, which temporarily coincides with an elevated
nutrient supply by the mother plant (source leaf ). However,
the question remains why not all seeds have green tissues. The
fact that this is not universal may suggest that under certain
conditions such a facility may be maladaptive. It can only be
speculated under which environmental circumstances seed
photosynthesis would have negative consequences. Similarly,
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Table 1 Experimental evidence for hypoxic limitations in seeds
Process
Major findings
Species: Reference
Assimilate uptake
Nutrient uptake by seed/embryo is limited by low O2;
possibly related to restrictions in energy-demanding
transport processes and/or sink activity
Low O2 restricts biosynthetic fluxes towards storage
products, in particular oil/protein rather than starch
Soybean: Thorne (1982)
Wheat: van Dongen et al. (2004)
Storage activity
Mitochondrial respiration
Fermentation activity
Assimilate partitioning
Shift in energy–consuming
processes
Metabolite distribution
mRNA translation
Enzyme activity
(except COX)
Low O2 limits energy production; respiration increases
at elevated O2 supply; developmental or day:night
changes in internal O2 level are reflected in energy
charge of cells
Level of fermentation products/fermentative enzyme activity
decreases at increasing O2 supply; developmental changes
in internal O2 are inversely related to fermentation activity
O2 affects assimilate partitioning between endosperm and embryo
Examples are the switch from invertase- to sucrose
synthase-mediated cleavage of sucrose or the (developmental)
switch from ATP to pyrophosphate (PPi) consuming pathways
Pattern of ATP distribution reflects the metabolic status of seed
tissues, and is O2-responsive in both spatial and temporal manner
Deposition pattern of legumin protein (major storage protein in
legumes) but not its mRNA correlates to that of ATP; it indicates
post-transcriptional regulation of protein storage via local
ATP/O2 availability
O2 affects the activity of fatty acid desaturases (FAD2) and
storage oil composition; FAD2 can occur as low- and
high-affinity enzyme, most likely regulated by O2-dependent
protein phosphorylation (J. Martinez-Rivas, pers. com.);
O2 affects key enzymatic activities of lipid metabolism
Wheat: Gifford & Bremner (1981)
Rapeseed: Vigeolas et al. (2003)
Soybean: Rolletschek et al. (2005a)
Maize: Rolletschek et al. (2005b)
Rapeseed: Vigeolas et al. (2003)
Wheat: van Dongen et al. (2004)
Soybean: Rolletschek et al. (2005a)
Maize: Rolletschek et al. (2005b)
Pea: Rolletschek et al. (2003)
Rapeseed: Vigeolas et al. (2003)
Maize: Rolletschek et al. (2005b)
Arabidopsis: Gibon et al. (2002);
Baud & Graham (2006)
Rapeseed: Vigeolas et al. (2003)
Maize: Rolletschek et al. (2005b)
Soybean: Rolletschek et al. (2005a)
Pea: Borisjuk et al. (2003)
Rapeseed: Vigeolas et al. (2003)
Sunflower: Rolletschek et al. (2007)
COX, cytochrome C oxidase.
there seems not to be a clear connection between photosynthetic activity of the seed and the nature of its carbohydrate or
lipid reserves.
In addition to the influence of light, temperature can also
reversibly affect the oxygen status of the seed. In a study on
developing sunflower seeds, a negative relationship between
oxygen level and the external temperature was established
(Rolletschek et al., 2007). As the temperature was raised from
10 to 40°C, the endogenous oxygen concentration fell rapidly; at
temperatures > 40°C the mean oxygen level dropped below 0.2 µm
but was able to recover to its initial value when the temperature
was decreased again. Typically, high temperatures increase respiration, but decrease oxygen solubility in the cell sap. In principle,
rising temperatures can also increase oxygen diffusivity, but this
effect was found to be negligible (Ho et al., 2007). The relationship
of oxygen to temperature appears rather trivial, but it has
important implications for fatty acid composition of oilseeds.
VI. Gene expression and metabolism under low
oxygen
Developing seeds are metabolically highly active. The early
phase of development features meristematic growth, and the
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subsequent seed-filling phase is characterized by endoreduplication and the synthesis of storage products (Weber et al., 2005).
A high respiration rate, combined with a limited gas exchange
capability, results in oxygen depletion inside the seed (Figs 2,
3). The question that remains is whether low oxygen really affects
gene expression, mitochondrial respiration and overall (storage)
metabolism. The oxygen levels commonly observed in seed tissue
seem to be sufficient for mitochondrial cytochrome oxidase
(COX) activity, as this enzyme has a low Km (between 0.08–
0.16 µm; Hoshi et al., 1993; Millar et al., 1994). However,
there might be an intracellular oxygen gradient, thus the mean
oxygen level measured by the microsensor is likely not to be
the value within the mitochondrion. Additionally, it is unclear
whether these in vitro estimates of Km apply in vivo (see Section
VII). Thus, the internal steady-state oxygen concentration of
10 µm in rapeseed appears to be nonlimiting, but the oxygenconsuming respiratory flux may be affected.
Several studies have demonstrated that assimilate uptake,
respiratory and storage metabolism of developing seeds are
oxygen-limited in vivo; an overview is provided by Table 1. A
detailed understanding of the molecular mechanisms underlying metabolic adjustment within the seed to hypoxia is yet
to emerge, but it seems reasonable to assume that at least some
© The Authors (2009)
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Tansley review
of the mechanisms established in roots or tubers (Geigenberger,
2003; Koch, 2004) also take place in the seed. Transcript and
metabolite profiling applied to study the hypoxic response of
A. thaliana roots/seedlings (Klok et al., 2002; Liu et al., 2005)
have identified a significant downregulation of genes associated
with the maintenance of cell wall integrity and ion transport,
along with an upregulation of those involved in transcriptional
regulation, protein kinase activity, glycolysis and fermentation
in oxygen depleted situations. Several genes of the signal transduction network (Ca2+ or phytohormone-dependent) and
specific sequence motifs in co-expressed genes have also been
highlighted. Some transcript profiling has been applied to the
developing barley caryopsis (Sreenivasulu et al., 2006, 2008),
and these same profiling tools are now being applied to study
the hypoxic/hyperoxic response of seeds. From some preliminary
data, it appears that the transcription level of genes associated
with glycolysis and mitochondrial energy metabolism (tricarboxylic acid (TCA) cycle and electron chain) are regulated in
vivo by a low internal oxygen level (N. Sreenivasulu, pers.
comm.). This result is supported by proteomic studies of the
developing maize caryopsis, in which Méchin et al. (2007) noted
an increase in the expression of glycolytic at the expense of
TCA cycle enzymes as development progressed. Thus, there is
an indication that the TCA cycle is regulated via an oxygendependent mechanism, possibly driven by ATP demand (see
also Fernie et al., 2004).
An analysis of oxygen maps suggests that in vivo, oxygen
limitation applies differentially to particular tissues/regions of
the seed. For example, in barley, oxygen deficiency is more
pronounced in the central than in the peripheral regions of the
endosperm, while the pericarp does not suffer from hypoxia
(Fig. 2c). Thus it is particularly difficult to suggest a generalized
effect of low oxygen status upon gene expression and metabolism within the whole caryopsis. A second example relates to
the pea, in which there is a gradient of oxygen concentration,
between high concentration at the outer edge and low concentration in the centre of the embryo. The genes for legumins
(storage proteins) are expressed in both regions, but protein
deposition occurs first in the peripheral regions of the embryo,
where ATP levels are locally upregulated (Borisjuk et al., 2003).
Based on this topographical coincidence it has been argued that
the energy-demanding protein synthesis (translation of legumin
mRNA) underlies control by local ATP availability (Borisjuk
et al., 2003). Thus, oxygen limitation to protein storage is
thought to occur only in the inner regions of the pea embryo
while outer ones are actually not oxygen limited. We believe
that a proper understanding of hypoxic adjustments in gene
expression and metabolism requires a topographical approach,
which considers both the local histological characteristics of the
tissue and the local oxygen concentration.
A series of other considerations are important for accurate
study of metabolism/gene expression in seeds. First, anoxia does
probably not occur in vivo during seed development and care
must be taken not to assume that hypoxia (Liu et al., 2005)
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Review
and anoxia (Loreti et al., 2005) have the same effect on seed
physiology. Second, in vitro experiments (Sriram et al., 2004;
Alonso et al., 2007), involving isolated embryos bathed in a
nutrient solution, may successfully mimic the chemical composition of the endosperm vacuole or phloem sap, but do not
reproduce the in vivo oxygen level. Third, since in vivo, the seed
oxygen status in green seeds depends heavily on light, experiments
which set out to measure the influence of oxygen status on storage metabolism may produce controversial results if the light
conditions are different. Thus, for example, lipid biosynthesis
in rapeseed is stimulated by additional external oxygen supply
at low light intensity (300 µmol quanta m−2 s−1; see Vigeolas
et al., 2003) but not at a higher light level (1000 µmol quanta
m−2 s−1; see Goffman et al., 2005).
VII. Mechanisms for oxygen sensing and
balancing in seeds
Changes in environmental conditions (e.g. light) produce shortterm adaptive responses in the oxygen consumption of the
developing seed (Vigeolas et al., 2003; Rolletschek et al., 2005a).
Elevating the external oxygen supply can be balanced by
increasing mitochondrial respiration (van Dongen et al., 2004;
Rolletschek et al., 2005b). During seed development, the levels
of typical fermentation products such as lactate/ethanol remain
rather low, thus fermentation and anoxia are obviously avoided.
It may be that seeds are able to fine-tune their endogenous
oxygen level as well as the oxygen demand. If so this would
require mechanisms for oxygen sensing and balancing.
Major insights into the molecular mechanisms of low-oxygen
sensing and subsequent signalling cascades have been obtained
from studies of bacteria, yeast and mammals (for review see
López-Barneo et al., 2001; Wenger, 2002; Schumacker, 2003).
Multiple pathways have been elucidated for oxygen sensing/
signalling, for example the hypoxia-inducible factor (HIF1)
and its modification via prolyl hydroxylases in mammals or
haem-binding proteins in yeast. It has been hypothesized that
various oxygen sensing/signalling mechanisms are tuned to
particular oxygen levels, enabling a plastic cellular response to
hypoxia. In plants, direct oxygen sensors of this type capable of
detecting/reacting with oxygen and triggering a signalling cascade
have yet to be identified. Rather, there is evidence for indirect
oxygen sensing based on changes in cellular homeostasis (e.g.
pH, ATP). The signal transduction chain is proposed to involve
the GTPase rheostat, modulations in the cytosolic Ca2+ level,
Ca2+-related protein kinases, the production of reactive oxygen
species, phytohormone action and probably much more (for
review see Geigenberger, 2003; Bailey-Serres & Chang, 2005;
Bailey-Serres & Voesenek, 2008).
The small gaseous molecule nitric oxide (NO) is sensitive to
oxygen, and acts to inhibit respiration via its reversible binding
to COX (Yamasaki et al., 2001; Zottini et al., 2002). In mammals, NO is a key player in the oxygen signalling and balancing
process. Endogenous NO levels are oxygen-dependent, but
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26 Review
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Fig. 5 Model of oxygen balancing in seeds
based on the regulation of mitochondrial
activity via the nitric oxide (NO)–O2 ratio.
Under O2 defiency, both stability and
synthesis of NO increases. Inhibition of
mitochondrial electron transport via NO
lowers O2 consumption, avoids anoxia and
tends to increase O2 availability.
Concomitantly, ATP-consuming processes,
including storage activity, are repressed.
An increase O2 availability (e.g. via seed
photosynthesis) lowers the NO–O2 ratio,
suspending mitochondrial inhibition.
This increases O2 consumption and ATP
availability, and eventually promotes the
biosynthetic activity/seed growth. In such
a way, NO triggers oxygen balancing in
an autoregulatory manner.
NO also modulates oxygen consumption as well as oxygen
availability (Moncada & Erusalimsky, 2002; Hagen et al., 2003;
Gong et al., 2004). In plants, NO appears to be involved in a
range of developmental and physiological processes, including
flowering, stomatal closure and plant–pathogen interactions
(Lamattina et al., 2003; Lamotte et al., 2005; Courtois et al.,
2008; Wilson et al., 2008). We have recently proposed a role
for NO in oxygen sensing/balancing in plants similar to the
one it plays in mammals (Borisjuk et al., 2007; Benamar et al.,
2008). Increases in endogenous oxygen concentration were
shown to decrease endogenous NO levels, while lowering it
strongly enhanced the level of NO (Borisjuk et al., 2007). The
rise in endogenous NO concentration at low oxygen levels
confirmed earlier results (Dordas et al., 2003; Planchet et al.,
2005), but the response was much faster than expected. The
kinetics of NO accumulation determines the short-term adaptive
response to hypoxia. NO accumulation can be attributed to
both its de novo synthesis and an increase in its stability. Oxygen
shortage enhances the accumulation of nitrite (Botrel et al.,
1996). Nitrite provides a substrate for NO synthesis in seeds
(Borisjuk et al., 2007) and is used by mitochondria under
hypoxia as an alternative electron acceptor (Stoimenova et al.,
2007; Benamar et al., 2008). This capability may have been
retained over the course of mitochondrial evolution. In addition
the amount of endogenous NO: controls respiratory activity
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and oxygen consumption, possibly via a modulation of the Km
of COX for oxygen; balances the steady-state oxygen level
in the seed; regulates mitochondrial ATP production; and
controls a number of energy-demanding processes, including
storage activity (Borisjuk et al., 2007).
We suppose that NO mediates rapid and reversible oxygen
balancing in seeds, including adjustments to the global metabolism
in response to changing oxygen supply (Fig. 5). Its dynamic
nature is of particular significance because it allows for the maintenance of a steady-state oxygen level and avoids the risk of the
onset of anoxia. Nitric oxide has been shown to increase the
transcript level of alternative oxidase (AOX; Huang et al., 2002).
It has been argued that this acts against the intended reduction
of oxygen consumption under oxygen-limiting conditions.
However, AOX activity in seeds is low and its affinity for oxygen is much lower than that of COX. Thus, the NO-mediated
stimulation of oxygen consumption via AOX is probably rare
in vivo. Instead, the induction of AOX transcript/activity may
be important to prevent programmed cell death (Vanlerberghe et al., 2002) and/or the increased formation of reactive
oxygen species (ROS) following anoxic injury (Maxwell et al.,
1999).
Our current thinking is that NO is a key regulator of oxygen
homeostasis in plants, comparable to the central role of HIF1
in mammals (Wenger, 2002; Schumacker, 2003). There is
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Tansley review
evidence that NO mediates responses which comprise an essential part of the oxygen signalling pathway. Both NO and low
oxygen stress cause: first, a transient increase in the level of cyclic
GMP (Pfeiffer et al., 1994; Reggiani, 1997), which in turn affects
ion channel activity and gene expression (Wilson et al., 2008);
second, a transient increase in the cytosolic level of calcium;
and third, an increase in the activity of MAP kinases, which
presumably affects gene expression (Courtois et al., 2008). A
significant degree of crosstalk exists between the NO and ROS
signalling pathways. Reactive oxygen species are released under
conditions of hypoxia (Zago et al., 2006). Where NO is produced in excess and/or cannot be easily degraded, additional
regulatory cascades may be induced by NO (e.g. protein
S-nitrosylation; ethylene synthesis), resulting in cell damage or
programmed cell death. By such mechanisms, NO might be
involved in the induction of aerenchyma, which is formed in roots
and rhizomes upon low oxygen stress (Justin & Armstrong, 1987).
It has been proposed that the cycling of NO back to nitrate
via nonsymbiotic class-1 haemoglobins represents an alternative respiratory pathway (Igamberdiev et al., 2005). Haemoglobins oxidize NADH, a process that helps to maintain the
energy and redox homeostasis under oxygen shortage. Haemoglobins have a high affinity for oxygen, which ensures that
they remain oxygenated at extremely low oxygen levels, and thus
can fulfil their function even when COX is no longer active.
Hence they can remove excess NO which might otherwise be
detrimental to the cell. Finally, haemoglobins may help to
remove NO from mitochondria/cells upon re-oxygenation, and
thus indirectly contribute to the reversibility of NO action.
Altogether, these mechanisms may explain the beneficial effects
of nonsymbiotic haemoglobins under hypoxic stress (Hunt et al.,
2002).
VIII. Might low internal oxygen levels be
advantageous for seed development?
The low internal oxygen concentration in the seed has a profound effect on storage product deposition, with clear implications for potential strategies to increase seed biomass and protein/
oil yield of crops. The question is whether molecular approaches
can be employed to increase oxygen availability within the crop
seed. The most obvious strategy would be to promote the
photosynthetic capacity of the seed. This would not only deliver
additional oxygen towards interior regions of the seed, but also
stimulate the biosynthetic input of plastids for cell metabolism,
promote energy availability for biosynthesis and increase carbon
use efficiency (Rolletschek & Borisjuk, 2005). An alternative
way to increase the availability of oxygen in seeds could be to
reduce the endogenous level of NO by either downregulating
its biosynthesis or upregulating its degradation. Under such
conditions, the respiratory activity of the seed is thought to
increase (less inhibition of COX as a result of lower NO levels),
followed by higher energy supply and corresponding adjustments
of the biosynthetic machinery. Another approach could manipu-
© The Authors (2009)
Journal compilation © New Phytologist (2009)
Review
late gas diffusivity by targeting cuticles or the intercellular space
(porosity). However, the identification of molecular targets for
such complex traits seems rather difficult.
Notably, a series of considerations would argue against
approaches to increase oxygen availability. The following sideeffects of increasing oxygen levels in the seed can be predicted:
• Increasing oxygen supply may promote seed biomass or oil
yield, but impairs carbon conversion efficiency (Goffman et al.,
2005), probably by inducing a higher respiration as well as a shift
in assimilate partitioning. This may be especially critical where
source capacity (supply with sucrose and amino acids) is limiting.
• The bioenergetic efficiency of mitochondria usually increases
at low oxygen levels (Gnaiger et al., 2000). Thus, the low internal
oxygen concentration present in the seed may promote the
carbon use efficiency.
• It may be advantageous to keep oxygen level low, to avoid
the formation of high levels of ROS (Simontacchi et al., 1995),
which damage cellular structures, requiring energy investment
for repair. In maize, the expression level of the genes associated
with detoxification (glutathione S-transferase, superoxide dismutases, ascorbate peroxidases) decreased during seed development (Méchin et al., 2007), consistent with the fall in oxygen
availability.
• Low internal oxygen levels in combination with high internal
CO2 levels (Wager, 1974a; Goffman et al., 2004) would minimize photorespiration. In such a way, the assimilate use efficiency is thought to increase.
• Nature has generated cuticles and barriers, which may reduce
oxygen uptake, but also help to prevent carbon dioxide loss.
Carbon dioxide levels in seeds are high, so a low gas exchange
capacity is helpful to promote the refixation of internally released
carbon dioxide that would otherwise escape from the seed.
Thus the carbon economy of seeds can be considerably improved
(Schwender et al., 2004).
• The cuticle acts as a barrier against water loss, pathogen
invasion and UV penetration. Weakening the barrier may compromise some of these essential functions. The deposition of
an intact cuticle may be a prerequisite for proper development
(Pruitt et al., 2000).
Overall, it may be advantageous for the seed to restrict its
gas exchange. Even though the internal oxygen concentration
falls to levels limiting respiration/storage metabolism, other
considerations also come into play. It remains to be seen whether
attempts to increase oxygen availability will result in higher
seed biomass and storage capacity in vivo, or whether some or
all of the above-mentioned factors act to negate any gain in
crop productivity. A comprehensive understanding of the
metabolism, structure and development of the seed is a prerequisite for initiating such approaches.
Acknowledgements
We thank Prof. U. Wobus for continuous support. We also
thank all scientists who contributed to this work, especially
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27
28 Review
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M. Hajirezaei, R. and V. Radchuk, T. Rutten, N. Sreenivasulu
and H. Tschiersch (Germany), D. Macherel and A. Benamar
(France), Bart Nicolai and Peter Verboeven (Belgium), Ivo
Feussner and Cornelia Göbel (Germany), Karen Koch (USA),
M. Mancha and J. Martinez-Rivas (Spain) and William
Armstrong (UK). We also thank U. Tiemann and K. Lipfert
for artwork. This work was supported by the Deutsche
Forschungsgemeinschaft.
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