Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.     

Phloem metabolism and function have to cope with low internal oxygen

We have investigated the consequences of endogenous limitations in oxygen delivery for phloem transport in Ricinus communis. In situ oxygen profiles were measured directly across stems of plants growing in air (21% [v/v] oxygen), using a microsensor with a tip diameter of approximately 30 microm. Oxygen levels decreased from 21% (v/v) at the surface to 7% (v/v) in the vascular region and increased again to 15% (v/v) toward the hollow center of the stem. Phloem sap exuding from small incisions in the bark of the stem was hypoxic, and the ATP to ADP ratio (4.1) and energy charge (0.78) were also low. When 5-cm stem segments of intact plants were exposed to zero external oxygen for 90 min, oxygen levels within the phloem decreased to approximately 2% (v/v), and ATP to ADP ratio and adenylate energy charge dropped further to 1.92 and 0.68, respectively. This was accompanied by a marked decrease in the phloem sucrose (Suc) concentration and Suc transport rate, which is likely to be explained by the inhibition of retrieval processes in the phloem. Germinating seedlings were used to analyze the effect of a stepwise decrease in oxygen tension on phloem transport and energy metabolism in more detail. Within the endosperm embedding the cotyledons-next to the phloem loading sites-oxygen decreased from approximately 14% (v/v) in 6-d-old seedlings down to approximately 6% (v/v) in 10-d-old seedlings. This was paralleled by a similar decrease of oxygen inside the hypocotyl. When the endosperm was removed and cotyledons incubated in a 100 mM Suc solution with 21%, 6%, 3%, or 0.5% (v/v) oxygen for 3 h before phloem sap was analyzed, decreasing oxygen tensions led to a progressive decrease in phloem energy state, indicating a partial inhibition of respiration. The estimated ratio of NADH to NAD(+) in the phloem exudate remained low (approximately 0.0014) when oxygen was decreased to 6% and 3% (v/v) but increased markedly (to approximately 0.008) at 0.5% (v/v) oxygen, paralleled by an increase in lactate and ethanol. Suc concentration and translocation decreased when oxygen was decreased to 3% and 0.5% (v/v). Falling oxygen led to a progressive increase in amino acids, especially of alanine, gamma-aminobutyrat, methionine, and isoleucine, a progressive decrease in the C to N ratio, and an increase in the succinate to malate ratio in the phloem. These results show that oxygen concentration is low inside the transport phloem in planta and that this results in adaptive changes in phloem metabolism and function.     

Manipulation of sucrose phloem and embryo loading affects pea leaf metabolism,carbon and nitrogen partitioning to sinks as well as seed storage pools

Seed development largely depends on the long‐distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source‐to‐sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this ‘Push‐and‐Pull’ approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source‐to‐sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1‐overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild‐type plants. Together, the results demonstrate that the SUT1‐overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push‐and‐Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.     

Phosphoenolpyruvate carboxykinase plays a role in interactions of carbon and nitrogen metabolism during grape seed development

Phosphoenolpyruvate carboxykinase (PEPCK) was shown to be present in a range of developing seeds by measurement of its activity and by immunoblotting. Its function was investigated during grape (Vitis vinifera L.) seed development. The maximum abundance of PEPCK coincided with the deposition of storage reserves. At this stage of development, immunohistochemistry showed that PEPCK was very abundant in a layer of cells located at the boundary of developing storage tissues and in the chalaza (close to the termination of the vascular supply to the seed) and was also present in the palisade layer of the seed coat (the inner layer of the outer integument). Earlier in development PEPCK was also present in the developing palisade layer and in the inner region of the nucellus which surrounds the developing endosperm. At later stages of development, PEPCK was located in the outer region of the endosperm. However, PEPCK was present in the phloem of the seed at all stages of development. Feeding of asparagine to developing grape seeds led to a strong induction of PEPCK. We suggest that, in developing grape seeds, both the chalaza and palisade tissue may distribute imported assimilates from the vasculature to the developing storage tissues and that PEPCK may play a role in the metabolism of nitrogenous assimilates during their delivery from the vasculature to the storage tissues. Received: 22 April 1999 / Accepted: 8 July 1999     

Phloem unloading and ‘sink strength’: The parallel between the site of attachment of Cuscuta and developing legume seeds

Sink regions play a central role in determining assimilate distribution patterns. Two factors are discussed which have a strong effect on the sink strength of a sink, viz. phloem unloading and turgor-sensitive transport. Sink strength may be defined as the capacity of phloem in the sink region to import assimilates from other parts of the plants and to release the imported substances into the sink apoplast.A stem parasitized by Cuscuta represents a very strong sink. A review is presented of data on enhanced phloem unloading, at the site of attachment of Custuta. Recent data on metabolically controlled sucrose and amino acid unloading into the seed coat apoplast of developing legume seeds show a remarkable parallel with phloem unloading in a parasitized Vicia faba stem. Data on turgor-sensitive sucrose and amino acid transport into developing seeds are presented, which throw new light on the pressure flow theory of phloem transport.     

Translocation in Plants Possessing Supernumerary Phloem: T. 14C-ASSIMILATES AND AUXIN IN THE INTERNAL PHLOEM OF TO BACCO (NICOTIANA TABACUML.)

Tobacco plants were exposed to ¹⁴CO₂ or treated with [¹⁴C]IAAin order to study the transport capacity of the internal phloem.It is shown that the translocation of assimilates in both theinternal as well as the external phloem is controlled by a source-sinkrelationship. The internal phloem is of a lesser conductiveimportance in the intact plant whereas in the girdled stemsit is of main importance as conductor. Auxin and other materialscontrolling apical dominance can also be translocated downwardsin the internal phloem in a sufficient amount in order to keepaxillary buds suppressed. Exogenous auxin applied to decapitatedstem can also be transported via the internal phloem and bypassgirdles, thus maintaining apical dominance.     

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.     

Increased phloem transport of S-methylmethionine positively affects sulfur and nitrogen metabolism and seed development in pea plants

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.     

Interaction of flooding with carbon metabolism of forest trees

Waterlogging and flooding cause oxygen deprivation in the root system of trees. Since oxygen is essentially for mitochondrial respiration, this process cannot be maintained under anoxic conditions and must be replaced by other pathways. For the roots it is therefore a matter of survival to switch from respiration to alcoholic fermentation. Due to the low efficiency of this process to yield energy equivalents (ATP), energy and carbon metabolism of trees are usually strongly affected by oxygen deprivation, even if a rapid switch from respiration to fermentation is achieved. The roots can compensate for the low energy yield of fermentation either (1) by decreasing the demand for energy by a reduction of energy-dependent processes such as root growth and/or nutrient uptake, or (2) by consuming more carbohydrates per unit time in order to generate sufficient energy equivalents. In the leaves of trees, flooding and waterlogging cause a decline in the rates of photosynthesis and transpiration, as well as in stomatal conductance. It is assumed that, due to reduced phloem transport, soluble sugars and starch accumulate in the leaves of flooded trees, thereby negatively affecting the sugar supply of the roots. Thus, root growth and survival is negatively affected by both changes in root internal carbon metabolism and impaired carbon allocation to the roots by phloem transport. In addition, accumulation of toxic products of fermentation in the roots, such as acetaldehyde, can further impair root metabolism. A main feature of tolerance against flooding and waterlogging of trees seems to be the steady supply of carbohydrates to the roots in order to maintain alcoholic fermentation; in addition, roots of tolerant trees seem to avoid accumulation of fermentation-derived ethanol and acetaldehyde. From studies with flooding tolerant and non-tolerant tree species, it is hypothesized that (1) the transport of ethanol produced in the roots under hypoxic conditions into the leaves via the transpiration stream, (2) its conversion into acetyl-CoA in the leaves, and (3) its use in the plant's general metabolism, are mechanisms of flooding tolerance of trees.     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Assimilate partitioning to and within attached seed coats.

The significance of the osmotic potential of the seed apoplast sap as a regulator of assimilate transfer to and within coats of developing seed of Vicia faba (cv. Coles Prolific) was assessed using attached empty seed coats and intact developing seed. Following surgical removal of the embryos, through windows cut in the pod walls and underlying seed coats, the resulting attached “empty” seed coats were filled with solutions of known osmotic potentials (–0. 02 versus –0. 75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (high osmotic concentration) for the first 190 min of exchange. Thereafter, this efflux was depressed relative to efflux from coats exposed to the low osmotic potential (high osmotic concentration) solution. This subsequent reversal in efflux was attributable to an enhanced diminution of the coat sucrose pools at the high external osmotic potential. Indeed, when expressed as a proportion of the current sucrose pool size, relative efflux remained elevated for coats exposed to the high osmotic potential solution. Measurement of potassium and sucrose fluxes to and from their respective pools in the coat tissues demonstrated that the principal, fluxes, sensitive to variative in the external osmotic potential, were phloem import into and efflux from the “empty” coats. Phloem import, consistent with a pressure-driven phloem transport mechanism, responded inversely with changes in the external osmotic potential. In contrast, sucrose and potassium efflux from the coats exhibited a positive dependence on the osmotic potential. Growth rates of whole seed were approximately doubled by enclosing selected pods in water jackets held at temperatures of 25°C. compared to 15°C. The osmotic potential of sap collected from the seed apoplast remained constant and independent of the temperature-induced changes in seed growth rates and hence phloem import. Based on these findings, it is proposed that control of phloem import by changes in the external osmotic potential observed with “empty” seed coats has no significance as a regulator of assimilate import by intact seed. Rather, maintenance of the seed apoplast osmotic potential, independent of seed growth rate, suggests that the observed osmotic regulation of efflux from the coats may play a key role in integrating assimilate demand by the embryo with phloem import.     

Oxygen balance for small organisms: an analytical model

An analytical model is developed that describes oxygen transport and oxygen consumption for small biological structures without a circulatory system. Oxygen inside the organism is transported by diffusion alone. Oxygen transfer towards the organism is retarded by a thin static fluid film at the surface of the organism. The thickness of this film models the outward water conditions, which may range from completely stagnant water conditions to so-called well-stirred water conditions. Oxygen consumption is concentration-independent above a specified threshold concentration (regulator behaviour) and is proportional to the oxygen concentration below this threshold (conformer behaviour). The model takes into account shape and size of the organism and predicts the transition from (pure) regulator behaviour to (pure) conformer behaviour, as well as the mean oxygen consumption rate. Thereby the model facilitates a proper analysis of the physical constraints set on shape and size of organisms without an active internal oxygen transport mechanism. This analysis is carried out in some detail for six characteristic shapes (infinite sheet, cylinder and beam; finite cylinder, sphere and block). In a well-stirred external medium, a flattened shape appears to be the most favourable for oxygen supply, while a compact shape (cube) is more favourable if the external medium is nearly stagnant. The theoretical framework is applied to oxygen consumption data of eight teleost embryos. This reveals relative insensitivity to external flow conditions in some species (e.g., winter flounder, herring), while others appear to rely on external stirring for a proper oxygen supply (e.g., largemouth bass). Interestingly, largemouth bass is the only species in our analysis that exhibits ‘fin-fanning’.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations.With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Abscisic acid and assimilate partitioning to developing seeds. II. Does abscisic acid influence the sink strength of Arabidopsis seeds?

The role of abscisic acid (ABA) in the regulation of transport of assimilates to seeds was investigated with the aid of Arabidopsis thaliana mutants that were ABA-deficient and/or insensitive to ABA. Subsequent flowers of mutant mother plants were alternately pollinated with pollen from either wild-type or mutant plants, and the transport of radiolabelled photoassimilates to the genetically different seeds was studied. The experiments were performed under conditions of reduced availability of source material, achieved either by reduced light quantity or by combining the ABA-deficient mutant with a starchless mutant. No effect of the genotype on the import rate of assimilates was detected, indicating that endogenous ABA does not influence the sink strength of Arabidopsis seeds. Reports describing contrary results are discussed.     

Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli

The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded beta-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased beta-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of beta-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.     

Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules

Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In fruits containing 4–6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 m M ), medium (350, 400 or 500 m M ) or high (0.7 or 1 M ) concentration of sucrose and/or mannitol. In pulse-labelling experiments with sucrose and α-aminoisobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules.
In our experiments, 350 m M appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmolarity of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in the seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the "sink strength" of developing seeds is turgor-sensitive.     

Low oxygen sensing and balancing in plant seeds: a role for nitric oxide

Storage product accumulation in seeds of major crop species is limited by their low internal oxygen concentration. Adjustment of energy and storage metabolism to oxygen deficiency (hypoxia) in seeds is highly relevant for agriculture and biotechnology. However, the mechanisms of low-oxygen sensing and balancing remain a mystery. Here, it is shown that normal hypoxia in seeds of soybean (Glycine max) and pea (Pisum sativum) triggers a nitrite-dependent increase in endogenous nitric oxide (NO) concentrations. NO, in turn, reduces the oxygen consumption of seeds, generating a localized decrease in both ATP availability and biosynthetic activity. Increasing oxygen availability reduces endogenous NO concentrations, thereby abolishing mitochondrial and metabolic inhibition. This auto-regulatory and reversible oxygen balancing, via NO, avoids seed anoxia and suggests a key role for NO in regulating storage activity. This hypothesis is reinforced by changes in energy status (ATP:ADP ratio), steady-state metabolite concentrations and biosynthetic fluxes under NO treatment. The proposed mechanism of low-oxygen sensing and balancing in plants offers the prospect of a new field of study in crop biotechnology.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Legume embryos develop in a hypoxic environment

Specific morphological and biochemical characteristics of seeds can cause oxygen deficiency within maternal and embryonic tissues. In this study, optical sensors were used to measure O(2) profiles across developing seeds of Vicia faba and Pisum sativum and developmental and environmental modulations of internal O(2) levels were studied. In addition, the metabolic state of developing embryos was analysed by monitoring adenylate energy charge, adenylate nucleotides and the levels of nucleotide sugars. Within the seed coat O(2) concentration decreased sharply to approximately 3% towards the inner border. Lowest O(2) levels were detected within the endospermal cavity between the seed coat and embryo. It is probable that low seed coat permeability provides an hypoxic environment for legume embryo development. The O(2) concentration in embryonic tissue changed during development with the lowest levels in the early stages. Measured in darkness, the levels were below 3%, but increased upon illumination indicating that photosynthesis significantly contributes to internal O(2) levels. Only in very young embryos were ATP levels and energy charge low. Otherwise they were maintained at a constant higher value. ADP-glucose and UDP-glucose did not show large fluctuations. Throughout embryo development fermentative activity did not play a major role. Obviously, specific mechanisms prevent seed tissues from becoming anoxic during development. The possible role of low oxygen on seed metabolism and on the control of seed development in legumes is discussed.