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 [14 C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14 C] 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 [15 N] 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.Legume seeds are a major source of plant-derived proteins and economically important for worldwide feed and food. Vicia and pea (Pisum sativum) seeds contain globulin storage proteins, hexameric legumins, and trimeric vicilins/convicilins, which together account for the majority of seed protein. The remainder consists of albumins, including lectins, lipoxygenases, proteinase inhibitors, late embryogenesis abundant proteins, and many other soluble proteins (Casey et al., 1993). Storage protein accumulation in legumes occurs in the embryo during maturation. Gln and/or Asn are translocated through the phloem (Miflin and Lea, 1977) and are symplastically unloaded into the seed coat where they are metabolized and reconstructed (Rochat and Boutin, 1991;Lanfermeijer et al., 1992). Mainly Gln, Ala, and Thr are released from the pea seed coat (Lanfermeijer et al., 1992) and, at maturation, Asn is also unloaded (Rochat and Boutin, 1991). Efflux of amino acids (and Suc) from pea seed coats is passive with linear kinetics, probably mediated by nonselective pores (DeJong et al., 1996(DeJong et al., , 1997. Amino acid uptake into soybean (Glycine max) and pea embryos is partially passive, especially during the early stage...

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.

The organ specificity of four promoters that are known to direct seed-specific gene expression was tested. Whereas the phaseolin (phas)- and legumin B4 (leB4)-promoters were from genes encoding 7S and 11S globulins from Phaseolus vulgaris and Vicia faba, respectively, the usp- and the sbp-promoters were from non-storage protein genes of V. faba. The expression of different promoter-reporter gene fusions was followed either by RT-PCR or by registering the reporter enzyme activity in organs of transgenic tobacco, pea, narbon bean, or linseed. In addition to seeds, the promoters directed reporter gene expression in pollen and in seed coats. USP-, vicilin- and legumin-mRNA were detected by RT-PCR in pollen of Pisum sativum and V. faba. Expression during microsporogenesis and embryogenesis seems to be a general character of various seed protein genes.

A full-length cDNA clone representing the large (shrunken-2) subunit of ADP-glucose pyrophosphorylase (AGP; EC 2.7.7.27) has been isolated from a cDNA library prepared from developing grain of hexaploid wheat (Triticum aestivum L., cv. Chinese Spring). The 2084-bp cDNA insert contains an open reading frame of 1566 nucleotides and primer-extension analysis indicated that the 5' end is 10 nucleotides shorter than the mRNA. The deduced protein contains 522 amino acids (57.8 kDa) and includes a putative transit peptide of 62 amino acids (6.5 kDa). The similarity of the deduced protein to the small subunit of AGP and to other AGP genes from plants and microorganisms is discussed. Northern hybridisation shows that the Agp1 genes (encoding the small subunit in the wheat endosperm) and the Agp2 genes (encoding the large subunit in the wheat endosperm) are differentially expressed in the wheat grain. Transcripts from both gene sets accumulate to high levels in the endosperm during grain development with the majority of the expression in the endopsperm rather than the embryo and pericarp layers. Although enzyme activity is detected in developing grains prior to 10 d post anthesis, only the Agp1 genes are active at this time (the Agp2 genes are not expressed until 10 d post anthesis). The possibility that the enzyme expressed during early grain development is a homotetramer of small subunits is discussed. The Agp1 and Agp2 genes are arranged as triplicate sets of single-copy homoeoloci in wheat. The Agp2 genes are located on the long arms of chromosomes 1A, 1B and 1D, about 80 cM from the centromere. The Agp1 genes have been mapped to a position just distal to the centromere on the long arms of chromosomes 7A, 7B and 7D.