To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUTl and VfSTPl, respectively. Sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUTl and VfSTP 7 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTPl mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUTl transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT7 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

Copy-DNA clones encoding a single class of sucrose-synthase (SUCS; EC 2.4.1.13) subunit have been isolated and sequenced from a Vicia faba L. seed cotyledonary library. Southern analyses indicated the existence of only one gene. Transcript levels determined by Northern blot hybridisation steadily increased until the middle of development [25-35 days after flowering (DAF)] and declined thereafter. Sucrose levels approximately paralleled levels of SUCS mRNA. The activity of SUCS increased with decreasing fructose and glucose concentrations and peaked about 10 d later than mRNA levels. In-vitro culture experiments demonstrated that increasing the sucrose concentration leads to increased levels of SUCS mRNA. The SUCS mRNA was also synthesised in seed-coat tissue, but in lower amounts than in cotyledons and with a different developmental profile. The early peak level of SUCS mRNA (20 DAF) in seed coats coincided with the peak in the amount of sucrose and with a peak of transiently synthesised starch.

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 have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated.

SummaryFull length cDNAs encoding three amino acid permeases were isolated from seed-speci®c libraries of Vicia faba. The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves. Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids. However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine. VfAAP1 was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues. Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter ful®lls an important role in providing amino acids for storage protein biosynthesis. VfAAP3 was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages. VfAAP4 transcripts could not be detected by northern hybridization. In situ hybridization showed that VfAAP1 mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer. It also accumulate in the chlorenchyma and thin-walled parenchyma cells of seed coats. VfAAP1 mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up-regulated under similar conditions. Cysteine repressed the expression of the GUS reporter gene under control of the VfAAP1 promoter, suggesting that this transporter is modulated at the transcriptional level. Regulation of amino acid transport in relation to storage protein accumulation is discussed.