Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Seed-specific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy

An ambitious aim in plant breeding and biotechnology is to increase the protein content of crop seeds used for food and feed. Using an approach to manipulate assimilate partitioning, we succeeded in elevating the protein content in legume seeds up to 50%. Transgenic bean plants were generated which express a Corynebacterium glutamicum phosphoenolpyruvate carboxylase (PEPC) in a seed-specific manner. The bacterial enzyme was not feedback inhibited by malate. Transgenic seeds showed a higher [14C]-CO2 uptake and about a threefold increased incorporation of labelled carbon into proteins. Changed metabolite profiles of maturing cotyledons indicated a shift of metabolic fluxes from sugars/starch into organic acids and free amino acids. These changes were consistent with an increased carbon flow through the anaplerotic pathway catalysed by PEPC. Consequently, transgenic seeds accumulated up to 20% more protein per gram seed dry weight. Additionally, seed dry weight was higher by 20%-30%. We conclude that PEPC in seeds is a promising target for molecular plant breeding.     

Differentiation of legume cotyledons as related to metabolic gradients and assimilate transport into seeds

Legume seed development is closely related to metabolism and nutrient transport. To analyse this relationship, a combination of biochemical, histological and transgenic approaches was used. Sugars within tissue sections have been quantitatively measured by metabolic imaging. During cotyledon differentiation glucose gradients emerge related to a particular cell type, with higher concentrations in non-differentiated premature regions. Sucrose in creases at the beginning of maturation in a layer underneath the outer epidermis expressing a sucrose transporter. Sucrose distribution is initially controlled by uptake activity and the permeability within the parenchyma and, later on, also by differences in growth and starch accumulation. Increased sucrose levels are accompanied by increased levels of sucrose synthase and ADP-Glc pyrophosphorylase mRNAs, but carbon flux into starch is initially still low. Rates increase at a stage when hexose concentrations become low, allowing increased flux through the sucrose synthase pathway. Transfer cell formation represents a regional specification of the cotyledonary epidermis for embryo nutrition characterized by increased transport-active cell surfaces and up-regulated expression of transport-related genes. The E2748 pea seed mutation blocks epidermal differentiation into transfer cells and leads to the loss of epidermal cell identity. Embryos with impaired epidermis cannot tolerate elevated levels of sucrose and respond with disorganized growth. The E2748 gene product is required for transfer cell formation in developing cotyledons with no other function during plant growth. Seed coat permeability provides a hypoxic environment for embryo development. However, at maturity, seed energy supply is not limited indicating fundamental developmental and metabolic adaptations. Results from transgenic seeds show that altered expression of single genes induces complex and unexpected changes. In AGP-antisense seeds the block in starch synthesis leads to pleiotropic effects of water and nitrogen content and induces temporal changes in seed development.     

Response of sugar interconversion, starch and protein accumulation to supplied metabolites during pod filling in chickpea

Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.     

Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase

In this work we test the hypothesis that yield of rice ( Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGP large subunit sequence ( Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat stability, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10-15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.     

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.     

Differential response of carbon and nitrogen metabolism to fluoride application in fruiting structures of chickpea (Cicer arietinum)

The effect of sodium fluoride (10 and 50 mol·m⁻³) on the activities of sucrose metabolizing enzymes, transaminases and glutamine synthetase in relation to the transformation of free sugars to starch and protein in the fruiting structures (pod wall, seed coat, cotyledons) of chickpea was studied by culturing detached reproductive shoots in a liquid medium. Addition of fluoride to the culture medium drastically reduced starch content of the cotyledons and caused a marked build-up of total free sugars comprised mainly of reducing sugars in the pod wall and seed coat, and sucrose in the cotyledons. Concomitantly, the activity of soluble invertase was stimulated in the pod wall but reduced in the cotyledons. However, soluble protein content of both the pod wall and the cotyledons increased in conjunction with an increase in the activities of glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and glutamine synthetase. Disruption of starch biosynthesis under the influence of fluoride and the resulting accumulation of free sugars possibly resulted in their favoured utilization in nitrogen metabolism. Labelling studies with [U-¹⁴C]-sucrose showed that the ¹⁴C incorporation into total free sugars was enhanced by fluoride in the pod wall but reduced in the seed coat and cotyledons, possibly due to an inhibitory effect on their translocation to the developing seeds.     

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.     

Sugar retrieval by coats of developing seeds of Phaseolus vulgaris L. and Vicia faba L

Influxes of glucose, fructose and sucrose were characterised for coat cells of developing seeds of Phaseolus vulgaris L. and Vicia faba L. by monitoring uptake of [(14)C]sugars into excised seed-coat halves and two different protoplast populations derived from seed coats. Sugar influxes by the two populations of protoplasts were similar for each sugar species [sucrose > (fructose approximately glucose)] and hexoses competed with sucrose. Concentration-dependent influxes of all three sugars by excised seed coats could be described by a simple directly proportional relationship between concentration ([S]) and uptake rate (v) in the physiological range of sugar concentrations (v approximately A.[S]). Alternatively, with the exception of fructose influx by Vicia, all could be fitted to a Michaelis-Menten relationship, as could sucrose uptake by Vicia protoplasts. Apparent K(m) values were high ( approximately 100-500 mM) compared with those reported for other systems. Sucrose transport was distinct from glucose and fructose transport in both species. Sugar influx was decreased by p-chloromercuribenzenesulfonic acid, carbonylcyanide m-chlorophenylhydrazone and erythrosin B. These responses are consistent with sugar/H(+) symport acting to retrieve photoassimilates leaked to the apoplasm during post-sieve element transport within seed coats.     

A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H⁺/hexose transporter and/or H⁺/sucrose symporter activities. Because SBP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SBP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide. In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SBP affects sucrose uptake in suspension-cultured cells.     

Sucrose synthase controls both intracellular ADP glucose levels and transitory starch biosynthesis in source leaves

The prevailing model on transitory starch biosynthesis in source leaves assumes that the plastidial ADPglucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule, ADPG. However, recent investigations have shown that ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol. This finding is consistent with the occurrence of an 'alternative' gluconeogenic pathway wherein sucrose synthase (SuSy) is involved in the production of ADPG in the cytosol, whereas both plastidial phosphoglucomutase (pPGM) and AGP play a prime role in the scavenging of starch breakdown products. To test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato wild-type (WT) leaves with those of the starch-deficient mutants with reduced pPGM and AGP. These analyses provided evidence against the 'classical' model of starch biosynthesis, since ADPG levels in all the starch-deficient lines were normal compared with WT plants. Whether or not SuSy is involved in the synthesis of ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and SuSy-antisensed transgenic leaves. Importantly, SuSy-overexpressing leaves exhibited a large increase of both ADPG and starch levels compared with WT leaves, whereas SuSy-antisensed leaves accumulated low amounts of both ADPG and starch. These findings show that (i) ADPG produced by SuSy is linked to starch biosynthesis; (ii) SuSy exerts a strong control on the starch biosynthetic process; and (iii) SuSy, but not AGP, controls the production of ADPG accumulating in source leaves.     

Embryo-specific reduction of ADP-Glc pyrophosphorylase leads to an inhibition of starch synthesis and a delay in oil accumulation in developing seeds of oilseed rape

In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting (14)C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of (14)C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation.