JXB Advance Access originally published online on April 8, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 401, pp. 1351-1359, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains
Received 28 November 2003; Accepted 18 February 2004
Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany
* To whom correspondence should be addressed. Fax: +49 39482 5500. E-mail: borysyuk{at}ipk-gatersleben.de
Abbreviations: AEC, adenylate energy charge; DPA, days post-anthesis; FW, fresh weight of tissue.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic profiling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the first time temporal and spatial maps of O2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained well-supplied with O2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, specific changes in the metabolite profiles (glycolysis and citrate cycle), and channelling of metabolic fluxes towards storage (increase of starch accumulation rate). These findings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level reflects the metabolic state of storage tissue. On the basis of these findings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.
Key words: Adenylate energy charge, ATP, barley seed, hypoxia, LC-MS, metabolite imaging, metabolite profiling, microsensor, tissue differentiation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
The agronomical importance of cereal seeds is based on their accumulation of storage products, mainly starch and proteins. Despite extensive studies on the structure, biochemistry, and genetics of developing grains (Duffus and Cochane, 1982; Olsen, 1992; Bewley and Black, 1994; James et al., 2003) the regulatory mechanisms underlying their high storage capacity are largely unknown. Storage product synthesis requires high amounts of energy provided by ATP and reducing equivalents. The level of adenine nucleotides affects storage because they function as co-substrates for many enzyme reactions, and may interact with protein kinases (Sugden et al., 1999). There is a growing body of evidence that biosynthesis in seeds is energy-limited (Neuhaus and Emes, 2000; Rolletschek et al., 2002a, 2003; Borisjuk et al., 2003). A control function of energy supply in storage was suggested. The amount of available energy can be represented by the adenylate energy charge (AEC; Pradet and Raymond, 1983). In barley, the AEC decreases during storage (Macnicol and Jacobsen, 1992) concomitant with the induction of fermentative enzymes (Macnicol and Jacobsen, 2001). From these findings, as well as somatic embryogenesis studies (Carman, 1998), it has been proposed that developing grains are hypoxic. This could have important implications for grain yield and quality. However, it still remains to be demonstrated that O2 levels inside the grain are low and likely to affect growth and storage.
Recently, it was demonstrated that legume seeds develop under hypoxic conditions (Rolletschek et al., 2002a). Embryo photosynthesis contributes significantly to oxygen supply and is coupled to biosynthetic fluxes (Rolletschek et al., 2003). ATP distribution within embryos is developmentally regulated and correlated with photosynthetic capacity (Borisjuk et al., 2003). In cereal grains, storage occurs in endosperm lacking any chlorophyll.
Until now, O2 and ATP-mapping of cereal grains has not been possible due their small size and complex morphology. Therefore, the general importance of conclusions drawn from legume seeds regarding hypoxia and energy state control of storage product biosynthesis remains to be tested.
Novel approaches are needed to analyse the spatial and temporal relationships of storage product accumulation, energy state, and O2 supply. In the work presented here, microsensors and a bioluminescence imaging technique allowed the spatial distribution of O2 and ATP in developing barley caryopses to be studied. For the first time, the existence of steep gradients in oxygen and ATP concentrations within the cereal grain are demonstrated. Their relation to tissue differentiation and starch storage pattern is illuminated. Based on the correlative analysis of these novel data, together with metabolite profiling and histological studies, a model is proposed, illustrating ideas about the possible role of oxygen and energy supply for storage patterning in barley grains.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Plant material
Barley plants (Hordeum vulgare cv. Barke) were cultivated in growth chambers under a light/dark regime of 16/8 h at 20/14 °C. Flowering ears were tagged for determination of days post-anthesis (DPA). Caryopses were harvested at distinct developmental stages in the mid-light/dark phase, and snap-frozen in liquid N2.
Determination of free amino acids, dissolved sugars, starch, pigments, and metabolites
Frozen plant material was weighed and homogenized by mortar and pestle. Free amino acids (FAA) and dissolved sugars were measured after ethanolic extraction by HPLC (Rolletschek et al., 2002a, b). Starch was measured photometrically as in Borisjuk et al. (2003). Chlorophyll was measured photometrically after extraction with acetone as in Borisjuk et al. (2003). For the determination of other metabolites plant material was extracted with trichloroacetic acid (Rolletschek et al., 2002a). Nucleotides and their sugars were determined by HPLC (Rolletschek et al., 2002a). Metabolites of gylcolysis and the citrate cycle were measured by ion chromatography coupled to mass spectrometry (IC-MS). This allowed the separation of the analytes depending on their retention times and their molecular masses, and the execution of parallel quantitative determinations with low detection limits. Chromatography was performed using a DX-600 ion chromatography system (Dionex, USA). Separation was carried out on a Dionex AS11-HC column (2x250 mm) and an additional guard column (AG11-HC) at 25 °C. A binary gradient at a constant flow rate of 0.5 ml min–1 was applied using distilled water (eluent A) and 100 mM sodium hydroxide (eluent B). The gradient was produced by the following linear concentration changes: 8 min 4% B, 20 min 30% B, 10 min 60% B, 10 min 100% B, hold 100% B for 3 min,; Return to 4% B in 3 min, and equilibrate for 10 min. Column effluents were directed to an ASRS-ULTRA (2 mm) 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 directed into the electrospray chamber, where ionization of separated compounds at atmospheric pressure took place. MS analysis was performed using a single quadrupole (MSQ, Dionex) with enhanced low mass option. The following parameters were employed: probe temperature 400 °C, sheath gas nitrogen, capillary voltage 3.5 kV, detection in negative ion mode using cone voltage of 50 V, and dwell time of 1 s. Up to 20 metabolites were measured in parallel using single ion monitoring (SIM) mode. Deprotonated ions [M-H]- were monitored with a span of 1 amu. Single SIMs were performed in small time windows of approximately 5 min during a total run time of 64 min. Up to five SIMs were run in parallel at maximum. This allowed parallel monitoring to be minimized and sensitivity to be enhanced, respectively. By comparing the results of IC-MS analyses with those from enzymatic assays it was possible to validate the concentration determined by IC-MS. In efforts to establish the efficiency of the whole extraction and measurement procedure, the recovery of metabolites was checked by the addition of metabolite standards to tissue samples (about 3-fold excess) at the start of extraction. Estimates of recovery were between 77% and 98%.
Measurement of internal O2 concentration
O2 concentration inside seeds was measured using O2-sensitive microsensors as described in detail earlier (Rolletschek et al., 2002a). Briefly, the intact caryopsis was carefully detached from the ear and fixed. Part of the palea (or lemma) were removed to allow correct positioning and to avoid destruction of the sensor (approximately 40 µm tip). This removal did not affect O2 levels within grains as it was checked upon initially. Subsequently, the sensor was driven into the seed by a micromanipulator. Measurments were done within 10 min. O2 concentration is given in percentages of atmospheric saturation (21 kPa=100%). Measurements were done in the light (about 700 µmol m–2 s–1) and darkness, respectively. After measurement, seeds were dissected at the measured transect to identify the exact position of the sensor.
Imaging of local ATP concentration
The method of imaging bioluminescence was used to measure ATP distribution in cryosections of caryopses. This technique allows the quantitative, histographical mapping of ATP and was described in detail earlier (Borisjuk et al., 2003). Briefly, cryosections were prepared from shock-frozen caryopses, and immersed into an enzyme solution linking ATP to the reaction of firefly luciferase. This enzyme emits photons with an intensity proportional to the content of ATP. The light emission was registered with a photon-counting system connected to a microscope. ATP concentrations determined by bioluminescence are considered in accordance with those determined by conventional HPLC. The high correlation coefficient (0.85; P <0.01) clearly indicates the validity of this method (Borisjuk et al., 2003).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
The onset of storage in endosperm is coupled with elevating starch accumulation rates and with an overall increase of metabolite levels
The single-seeded fruit known as a caryopsis (Fig. 1A) consists of the pericarp and testa surrounding the endosperm and the embedded embryo. During development, the pericarp degenerates, and the enlarging endosperm becomes the main storage organ (Fig. 1B, D). Starch is the main storage product in barley. Accordingly, the size and localization of starch grains indicate the topographical arrangement of storage and the starch accumulation rate can be used to characterize storage activity.
|
Seed development was analysed from 0–20 d post-anthesis (DPA), covering the prestorage, intermediate, and storage phases. Within the prestorage phase (0–5 DPA) the caryopsis consists mainly of pericarp tissue, embedding the liquid endosperm and the embryo. Increase of fresh weight and starch accumulation was low (Fig. 1F), the latter is mainly found in the pericarp (Weschke et al., 2000). Levels of glycolytic and citrate cycle metabolites were consistently low, but free amino acids (FAA) and soluble sugars started to increase (Fig. 2). The subsequent intermediate phase begins after endosperm cellularization at 4–5 DPA and proceeds with the differentiation of endosperm tissues. Starch accumulation starts with low synthesis rates (calculated from starch accumulation per day and per caryopsis; Fig. 1F). In parallel, a slight increase in several metabolites of glycolysis and the citrate cycle was also evident (Fig. 2). During the main storage phase (from 10–11 DPA onwards), a high starch synthesis rate was found indicating high storage activity. Rising fluxes into starch were coupled with a strong increase in ADP-glucose (Fig. 2), the direct precursor for the starch synthase reaction (James et al., 2003). All measured metabolites of the glycolytic and citrate cycle pathway increased similarly several-fold (Fig. 2). Both the flux into starch (starch synthesis rate) and metabolite levels peaked at approximately 14 DPA, and remained at high levels thereafter. In accordance with these metabolic changes, glycolytic and storage-associated genes, e.g. ADP-glucose pyrophosphorylase, were induced at the onset of storage (Sreenivasulu et al., 2004). The described changes imply that storage activity is coupled with elevated metabolic fluxes, reflected in higher metabolite levels.
|
Is the storage phase energy-limited?
Increasing metabolic activity (e.g. starch accumulation rate) during storage may affect energy status, and vice versa. In order to investigate this relationship, the adenine nucleotide pool was analysed (Fig. 3). During the prestorage phase the level of AMP, ADP, and ATP as well as the adenylate energy charge (AEC=(ATP+0.5x ADP)/(ATP+ADP+AMP) was fairly constant (Fig. 3A). Upon entering the storage phase there was an increase in all three adenine nucleotides. This reflects the overall change in metabolic activity at this time, and was also found for other metabolites (Fig. 2). However, the AEC started to decrease in the process of further development (main storage phase), mainly due to both lower ATP and higher AMP levels.
|
It was concluded from the data obtained that the main storage phase becomes increasingly energy-limited. This limitation can be regarded as a result of (i) the elevated metabolic activity that leads to an increased energy demand or/and (ii) the decrease of energy supply. These data showed high alanine and malate levels during storage (Fig. 2), suggesting fermentation activity (Macnicol and Jacobsen, 1992), and up-to 5-fold increases in the lactate to pyruvate ratio (data not shown) during the night, indicating a strongly reduced NAD+ system. It implicates an in vivo O2-limited respiration, and is also reflected by the induction of fermentation enzymes (Macnicol and Jacobsen, 2001).
These results seem to be at variance with the correlation of high energy and storage during Vicia and pea seed development which were described earlier (Rolletschek et al., 2003; Borisjuk et al., 2003). Therefore, the existence of a specific spatial arrangement of both storage activity and high energy levels was assumed within the developing endosperm, concealed by whole organ analysis. In order to prove this assumption, the spatial distribution of ATP was investigated.
ATP distribution is developmentally controlled
A recently developed quantitative bioluminescence method (Borisjuk et al., 2003) was applied for measurement of ATP concentrations directly in cryosections of the developing barley grain. The ATP pool is directly related to AEC by the activity of adenylate kinases. By plotting AEC versus ATP a near-linear correlation can be observed up to an AEC of about 0.9 (data not shown). A similar relationship was found for legume embryos (Borisjuk et al., 2003). Therefore, the steady-state level of ATP was chosen here as an indicator of the energy status of tissues. On the basis of the ATP distribution, the imaging technique allowed the ATP/energy state to be related to the developmental state of tissue. Representative ATP maps are shown in Fig. 3B–D and Fig. 5. In general, the tissues wrapped by the embryo sac, mostly endosperm, had much higher ATP levels than the pericarp and the nucellar projection. During the prestorage phase, ATP was slightly elevated in the caryopsis’ central region (Fig. 3B). During the intermediate phase, high ATP characterized the entire endosperm with the highest local levels (up to 1000 nmol g–1 FW) in the central regions of the lateral endosperm (cheeks), as can be seen on the ATP map in Fig. 5. Later on, the ATP-rich zone expanded to the ventral region connecting the two lateral parts like a bridge (Fig. 3C). During the storage phase, high levels of ATP became associated with the peripheral regions (Fig. 3D). ATP decreased within the central part of the endosperm. This low ATP-region expanded during development.
|
Simonds and O’Brien (1981) described in detail the asymmetric dorso-ventral growth pattern of the caryopsis. According to this pattern, a developmental gradient is formed within the endosperm: mitotically active cells mostly on the periphery, expanding starch-accumulating cells predominantly towards the median zone and, finally, fully differentiated storage cells in the central region. This gradient is reflected in cell size and the size of starch grains, and is recognizable during the entire development. The topographical relations of ATP distribution and histological pattern (Fig. 3E-H) were analysed. During the intermediate phase, high ATP (red colour in the ATP map, Fig. 3C) was correlated with storage cells containing a vacuole and multiple small starch grains (sc in Fig. 3E). ATP concentration declined towards the elongating cells (ec) and was minimal in small, mitotically-active peripheral cells (mc). Towards maturation, high ATP (Fig. 3D) became associated with aleurone (al in Fig. 3H), where the protein storage occurs (data not shown), and aleurone-attached starchy endosperm containing only small starch grains (ae). Low ATP was characteristic for fully differentiated starchy endosperm cells (se) already occupied by large starch grains. Thus, high ATP is characteristic for actively starch-accumulating cells. It is concluded that ATP distribution patterns are related to the differentiation of endosperm towards storage tissue. Thereby, a high ATP level is associated with the local onset of storage.
Oxygen supply to the endosperm is inherent in the chlorophyll pattern of the caryopsis
Within the endosperm, ATP synthesis via respiration depends on O2 supply. There is experimental evidence that O2 is generated in the pericarp by photosynthesis (Caley et al., 1990; Nutbeam and Duffus, 1978; Duffus and Cochrane, 1982). Photosynthetic activity is correlated with chlorophyll content (Evans and Rawson, 1970; Caley et al., 1990). Chlorophyll content increased within the intermediate growth phase (Fig. 4D) immediately after the induction of photosynthesis-related genes (Sreenivasulu et al., 2004). Using laser scanning microscopy, it can be shown that chlorophyll is located in a distinct layer within the pericarp, covering the whole endosperm except for the region of veins and nucellar projection (Fig. 1B, C). This pattern was maintained during the entire development (Fig. 4D, E). The existence of these chlorophyll layers may suggest that photosynthesis (O2 production) plays a role in grain growth.
|
In an effort to validate this suggestion, the internal O2 distribution under light and darkness was analysed using microsensors. During the prestorage phase, O2 declined from the outer pericarp layer towards its inner layers (Fig. 4A). Within the liquid endosperm O2 content was fairly low (about 1% of atmospheric saturation) and constant. During the early storage phase, sharp O2 gradients were observed in caryopses (Fig. 4B). Measured in the light, O2 increased within the pericarp up to 250% and peaked within the chlorophyll strands (±50–100 µm, red dotted line in Fig. 4). The peak O2 level was dependent on light intensity (data not shown). Towards the interior of the caryopses, the level of O2 fell sharply and was lowest within the central endosperm. During the late storage phase, an O2 peak was detectable only in the ventral chlorophyll layers (close to the nucellar projection). The low-O2 zone in the central region of the endosperm became dramatically enlarged during development (compare Fig. 4B and C). The minimum O2 level was dependent on the developmental stage: it decreased from about 2% at the 20 mg FW-stage to 0.6% at the 40 mg FW-stage. This indicates an increasing hypoxic strength during development, and corresponds to the induction of fermentation as described above.
Overall, the shape of the concentration gradients clearly indicates that (i) O2 is produced at high rates within the chlorophyll-containing layers of the pericarp, and (ii) O2 flows towards both the endosperm and the outer pericarp. Lateral and peripheral regions of endosperm are favourably supplied with O2. Oxygen supply to the endosperm is therefore inherent in the chlorophyll pattern. The sharp O2 decline within the endosperm during the storage phase points to a strong consumption due to high metabolic activity. Integuments attached to the endosperm (except at the creased vein area, cf. Zee and O’Brien, 1970) obviously allow O2 diffusion as derived from O2 concentration gradients. Nevertheless, they may impede gas exchange (Cochrane and Duffus, 1979). This would, in addition, account for sharp gradients. It was concluded further that the role of pericarp photosynthesis for the storage process consists more of oxygen supply to the endosperm then of CO2 fixation (only 2% of final starch are derived from grain photosynthesis; cf. Watson and Duffus, 1988).
Central regions of growing caryopses become strongly hypoxic
Within caryopses of the prestorage phase, oxygen concentrations were moderately low (Fig. 4A). There were no changes in O2 levels upon light/darkness (data not shown). This indicates the lack of photosynthetic activity at an early stage and corresponds to the chlorophyll profile (Fig. 4D). During the intermediate and early storage phases (Fig. 4E) the O2 levels in darkness fell below 0.1% within the nucellar projection cells/endospermal cavity/endospermal transfer cells. These tissues represent an essential part of the main transport route for assimilates (Patrick and Offler, 2001). During further development, the low O2 region spreads on the starchy endosperm (compare Fig. 4E and F). O2 levels decreased to near zero (<0.1%) and were below the resolution of the microsensor. Thus, about 10-fold differences in oxygen concentration within the central endosperm (1.2 versus <0.1 during light and darkness, respectively) were apparent. The O2 levels found here may still be sufficient for plant mitochondrial cytochrome oxidase due to its low Km (Drew, 1997). However, hypoxic responses are already observed at relatively high O2 levels (5–10%; Rolletschek et al., 2003; Vigeolas et al., 2003). Concomitant with lower oxygen levels in the caryopses at night, metabolic profiling showed changes characteristic for hypoxia: lower energy charge levels (Fig. 3A) and a decrease of glycolytic metabolites downstream of Fru1,6diP (Fig. 2) during the night.
Together, it can be shown that developing caryopses are becoming hypoxic during the intermediate and storage phases, especially during the night. Metabolic adaptations to low oxygen generally result in the down-regulation of energy-consuming processes, including storage activity. Our data strongly suggest that both nutrient transport to the endosperm and storage activity within the endosperm are affected by local oxygen depletion. Twenty-years ago, Gifford and Bremner (1981) could show that light stimulates in vitro the uptake and incorporation of 14C-sucrose into starch by wheat kernels, possibly via oxygen delivery by pericarp photosynthesis. It could further be shown that superambient oxygen levels boosted starch synthesis in the light (by 30% compared with ambient O2 levels), but, in particular, in darkness (over 5-fold). The oxygen and ATP maps can explain the findings by Gifford and Bremner, and allow the following conclusions: (i) storage is oxygen/energy-limited under in vivo conditions, and (ii) photosynthetic oxygen supply can be a strong determinant for storage product accumulation. Day/night changes in the metabolic activity of caryopses (as in Gifford and Bremner, 1981) can easily be explained as a response to varying oxygen levels inside the caryopsis. This would also be consistent with findings on light effects on metabolic and storage activity in seeds of other species (Vicia faba: Rolletschek et al., 2003; soybean: Willms et al., 1999; oilseed rape: Eastmond and Rawsthorne, 1998).
High ATP and storage activity occur in regions well supplied with oxygen
O2 concentration was analysed in relation to ATP distribution and storage pattern. Lateral regions of the endosperm, where storage activity is initiated, are surrounded by potosynthetically-active tissue. O2 supply in this region is most favourable (Fig. 5). In the groove region, O2 is supplied mostly from one (ventral) direction because vein, nucellar projection, and the endospermal cavity lack chlorophyll. In this region low O2 is found, and both ATP and starch accumulation are delayed temporally. High ATP concentrations and starch-accumulating cells are located along the O2-enriched, endospermal tissues (Fig. 5). This topographical correlation can be drawn from the comparison of oxygen concentration, the ATP map, and starch deposition within the caryopses (Fig. 5). During further development, accumulation zones of ATP and starch move towards peripheral regions (Fig. 3C–H) which are always O2-enriched. It was concluded that the endospermal storage activity, which is coupled with high energy demand, occurs in regions with favourable O2 supply. Under O2 limitation, this localization is crucial for respiratory ATP synthesis.
Beside ATP, organic substrates are needed for both respiration and storage activity. Sucrose can play a double role in starch storage: as a signal molecule and as a precursor for starch synthesis (Smith et al., 1997; Wobus and Weber, 1999; Rolland et al., 2002; Sun et al., 2003); the rate of starch accumulation is a function of sucrose concentration (Jenner et al., 1991). High levels of sucrose were shown to induce genes of the starch biosynthesis pathway (Koch, 1996). In barley, sucrose is the major sugar during grain-filling. Sucrose is associated with up-regulation of the sucrose transporter, induction of sucrose synthase, and the onset of storage (Weschke et al., 2000). Sucrose is delivered from the maternal tissue through the nucellar projection and taken up by the attached endospermal transfer cells (Patrick and Offler, 2001). Further distribution of sucrose occurs symplastically (Wang et al., 1995), driven by local sink activity within the peripheral regions of the endosperm. The consequent flow of sucrose is directed towards O2 flow. Because low O2 may limit ATP supply and low sucrose may limit starch synthesis, the availability of both of these metabolites can significantly influence the storage process. This idea about the possible interactions of oxygen distribution, carbohydrate supply, and energy state of the tissue within the developing caryopsis, is illustrated in Fig. 6. This hypothetical model suggests that optimal grain filling is achieved by sufficient oxygen supplied from the photosynthesizing pericarp towards the inner part of the endosperm, and by sugar flow towards peripheral regions of the endosperm. The zones that are favourably supplied with both sugars and oxygen, produce the highest amount of ATP and, accordingly, storage polymers. This would account for the pattern of starch accumulation observed during development: starch grains appear first in the central region of the lateral endosperm (intermediate phase), are than found along the median line in the groove, and finally the starch-accumulating zone migrates towards the periphery in later stages (cf. Simonds and O’Brien, 1981).
|
|
Concluding remarks |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
This study provides the first experimental evidence for oxygen and ATP gradients within cereal grains. Very low O2 levels were found inside grains well known to limit respiration and overall metabolic activity. During development, hypoxic regions expand and cover the major part of the starchy endosperm. The increasing oxygen depletion is reflected in lower energy state and changing metabolite pattern. Topographically, hypoxic regions were characterized by a locally-decreased steady-state level of ATP. High concentrations of ATP corresponded with metabolically active regions involved in storage biosynthesis. This temporal and spatial correlation maintained during development indicates that a high ATP/energy state is characteristic of actively-storing tissues within the barley endosperm. It is reasonable to assume that a high ATP/energy state might even be necessary to fuel the elevated metabolic fluxes. Further evidence comes from studies with legume seeds, where the energy state was related to the distribution of storage activity as well as to the partitioning of assimilates into different storage product classes (Borisjuk et al., 2003; Rolletschek et al., 2003). This suggests a general regulatory role of energy state on storage activity in seeds. Independent of their specific morpho-physiological features and photosynthetic capabilities, seeds have to cope with a limited oxygen and energy availability to maintain their high storage capacity. These findings lay the groundwork for further investigations, including molecular approaches (transgenic/reverse genetics).
The role of O2 and ATP may not be limited to its function as an electron acceptor in the respiratory chain and energy source, respectively. Both are also known as signal molecules. It has been reported that O2 acts in plants as a signal affecting gene expression (Drew, 1997; Klok et al., 2002). In the mammalian system ATP sensors were recently described (Dennis et al., 2001; Lópes-Barneo et al., 2001; Ryten et al., 2002). Plant sensors tuned to distinct O2/ATP levels, as well as signal cascades and transcription factors acting in this process, are currently unknown.
|
Acknowledgements |
|---|
We are grateful to Dr S Walenta and Dr M Rokitta for co-operation, and to U Tiemann, K Lipfert, B Claus, K Blaschek, and G Einert for excellent technical assistance. This work was supported by the Land Sachsen-Anhalt (MK-LSA 0031KL/1002L).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Bewley JD, Black M. 1994. Seeds: physiology of development and germination. New York: Plenum Press.
Borisjuk L, Rolletschek H, Walenta S, Panitz R, Wobus U, Weber H. 2003. Energy status and its control on embryogenesis of legumes. ATP distribution within Vicia faba embryos is developmentally regulated and correlated with photosynthetic capacity. The Plant Journal 36, 318–329.[CrossRef][Web of Science][Medline]
Caley CY, Duffus CM, Jeffcoat B. 1990. Photosynthesis in the pericarp of developing wheat grains. Journal of Experimental Botany 41, 303–307.
Carman JG. 1998. Improved somatic embryogenesis in wheat by partial simulation of the in ovulo oxygen, growth-regulator, and desiccation environments. Planta 175, 417–424.[CrossRef]
Cochrane MP, Duffus CM. 1979. Morphology and ultrastructure of immature cereal grains in relation to transport. Annals of Botany 44, 67–72.
Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G. 2001. Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105.
Drew MC. 1997. Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia. Annual Review of Plant Physiology and Plant Molecular Biology 48, 223–250.[CrossRef][Web of Science][Medline]
Duffus CM, Cochrane MP. 1982. Carbohydrate metabolism during cereal grain development. In: Khan AA, ed. The physiology and biochemistry of seed development, dormancy and germination. Elsevier Biomedical Press, 43–66.
Eastmond PJ, Rawsthorne S. 1998. Comparison of the metabolic properties of plastids isolated from developing leaves or embryos of Brassica napus. Journal of Experimental Botany 49, 1105–1111.
Evans LT, Rawson HM. 1970. Photosynthesis and respiration by the flag leaf and components of the ear during grain development. Australian Journal of Biological Sciencess 23, 245–254.
Gifford RM, Bremner PM. 1981. Accumulation and conversion of sugars by developing wheat grains. II. Light requirements for kernels cultured in vitro. Australian Journal of Plant Physiology 8, 631–640.
James MG, Denyer K, Myers AM. 2003. Starch synthesis in the cereal endosperm. Current Opinion in Plant Biology 6, 215–222.[CrossRef][Web of Science][Medline]
Jenner CF, Uglade TD, Aspinall D. 1991. The physiology of starch and protein deposition in the endosperm of wheat. Australian Journal of Plant Physiology 18, 211–226.
Klok EJ, Wilson IW, Wilson D, Chapman SC, Ewing RM, Somerville SC, Peacock WJ, Dolferus R, Dennis ES. 2002. Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. The Plant Cell 14, 2481–2494.
Koch KE. 1996. Carbohydrate-modulated gene expression in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 509–540.[CrossRef][Web of Science]
López-Barneo J, Pardal R, Ortega-Sáenz P. 2001. Cellular mechanisms of oxygen sensing. Annual Review of Physiology 63, 259–287.[CrossRef][Web of Science][Medline]
Macnicol PK, Jacobsen JV. 1992. Endosperm acidification and related metabolic processes in the developing barley grain. Plant Physiology 98, 1098–1104.
Macnicol PK, Jacobsen JV. 2001. Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid. Physiologia Plantarum 111, 533–539.[CrossRef][Medline]
Neuhaus HE, Emes MJ. 2000. Non-photosynthetic metabolism in plastids. Annual Review of Plant Physiology and Plant Molecular Biology 51, 111–140.[CrossRef][Web of Science]
Nutbeam AR, Duffus CM. 1978. Oxygen exchange in the pericarp green layer of immature cereal grains. Plant Physiology 62, 360–362.
Olsen OA, Potter RH, Kalla R. 1992. Histo-differentiation and molecular biology of developing cereal endosperm. Seed Science Research 2, 117–131.
Patrick JW, Offler CE. 2001. Compartmentation of transport and transfer events in developing seeds. Journal of Experimental Botany 52, 551–564.
Pradet A, Raymond A. 1983. Adenine nucleotide ratios and adenylate energy charge in energy metabolism. Annual Review of Plant Physiology 34, 199–224.[Web of Science]
Rolland F, Moore B, Sheen J. 2002. Sugar sensing and signaling in plants. The Plant Cell, Supplement, S185–S205.
Rolletschek H, Borisjuk L, Koschorreck M, Wobus U, Weber H. 2002a. Legume embryos develop in a hypoxic environment. Journal of Experimental Botany 53, 1099–1107.
Rolletschek H, Hajirezaei MR, Wobus U, Weber H. 2002b. Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars, causes higher uptake of water and amino acids which leads to higher protein content. Planta 214, 954–964.[CrossRef][Web of Science][Medline]
Rolletschek H, Weber H, Borisjuk L. 2003. Energy status and its control on embryogenesis of legumes: embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes. Plant Physiology 132, 1196–1206.
Ryten M, Dunn PM, Neary JT, Burnstock G. 2002. ATP regulates the differentiation of mammalian skeletal muscle by activation of a P2X5 receptor on satellite cells. Journal of Cell Biology 158, 345–355.
Simonds DH, O’Brien TP. 1981. Morphological and biochemical development of the wheat endosperm. Advances in Cereal Sciences and Technology 4, 5–70.
Smith AM, Denyer K, Martin C. 1997. The synthesis of the starch granule. Annual Review of Plant Physiology and Plant Molecular Biology 48, 67–87.[CrossRef]
Sreenivasulu N, Altschmied L, Radchuk V, Gubatz S, Wobus U, Weschke W. 2004. Transcript profiles and deduced changes of metabolic pathways in maternal and filial tissues of developing barley grains. The Plant Journal 37, 539–553.[CrossRef][Web of Science][Medline]
Sugden C, Crawford RM, Halford NG, Hardie DG. 1999. Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5'-AMP. The Plant Journal 19, 433–439.[CrossRef][Web of Science][Medline]
Sun C, Palmqvist S, Olsson H, Boren M, Ahlandsberg S, Jansson C. 2003. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by bindung to the sugar-responsive elements of the ios1 promoter. The Plant Cell 15, 2076–2092.
Vigeolas H, Dongen JT van, Waldeck P, Hühn D, Geigenberger P. 2003. Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape. Plant Physiology 133, 2048–2060.
Wang HL, Patrick JW, Offler CE, Wang XD. 1995. The cellular pathway of photosynthates transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm. Plant, Cell and Environment 18, 389–407.
Watson PA, Duffus CM. 1988. Carbon dioxide fixation by detached cereal caryopses. Plant Physiology 87, 504–509.
Weschke W, Panitz R, Sauer N, Wang Q, Neubohn B, Weber H, Wobus U. 2000. Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation. The Plant Journal 21, 455–467.[CrossRef][Web of Science][Medline]
Willms JR, Salon C, Layzell DB. 1999. Evidence for light-stimulated fatty acid synthesis in soybean fruit. Plant Physiology 120, 1117–1128
Wobus U, Weber H. 1999. Sugars as signal molecules in plant seed development. Journal of Biological Chemistry 380, 937–944.
Zee SY, O’Brien TP. 1970. Studies on the ontogeny of the pigment strand in the caryopsis of wheat. Australian Journal of Biological Sciencess 23, 1153–1171.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. V. Radchuk, L. Borisjuk, N. Sreenivasulu, K. Merx, H.-P. Mock, H. Rolletschek, U. Wobus, and W. Weschke Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain Plant Physiology, May 1, 2009; 150(1): 190 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Grafahrend-Belau, F. Schreiber, D. Koschutzki, and B. H. Junker Flux Balance Analysis of Barley Seeds: A Computational Approach to Study Systemic Properties of Central Metabolism Plant Physiology, January 1, 2009; 149(1): 585 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Xu, T. Li, Z. Y. Deng, K. Chong, Y. Xue, and T. Wang Dynamic Proteomic Analysis Reveals a Switch between Central Carbon Metabolism and Alcoholic Fermentation in Rice Filling Grains Plant Physiology, October 1, 2008; 148(2): 908 - 925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sreenivasulu, B. Usadel, A. Winter, V. Radchuk, U. Scholz, N. Stein, W. Weschke, M. Strickert, T. J. Close, M. Stitt, et al. Barley Grain Maturation and Germination: Metabolic Pathway and Regulatory Network Commonalities and Differences Highlighted by New MapMan/PageMan Profiling Tools Plant Physiology, April 1, 2008; 146(4): 1738 - 1758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rolletschek, L. Borisjuk, A. Sanchez-Garcia, C. Gotor, L. C. Romero, J. M. Martinez-Rivas, and M. Mancha Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds J. Exp. Bot., September 27, 2007; (2007) erm154v1. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||