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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Ectopic Expression of an Amino Acid Transporter (VfAAP1) in Seeds of Vicia narbonensis and Pea Increases Storage Proteins¹

Institut für Pflanzengenetik und Kulturpflanzenforschung, D–06466 Gatersleben, Germany (H.R., F.H., M.M., U.H., A.S., L.B., I.S., U.W., H.W.); and Fachgebiet Pflanzenbau in den Tropen und Subtropen, Humboldt Universität zu Berlin, D–14195 Berlin, Germany (K.-P.G.)

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.

² Present address: Biology Department, University of the West Indies, Trinidad.

³ Present address: Biology Department, University of Victoria, Victoria, British Columbia, Canada V8W 5N5.

⁴ Present address: Sungene, BASF Plant Science, D–06466 Gatersleben, Germany.

⁵ Present address: Max-Planck-Institut für Molekulare Pflanzenphysiologyie, D–14476 Golm, Germany.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056523.

^* Corresponding author; e-mail weber{at}ipk-gatersleben.de; fax 49–39482–5500.

Received November 13, 2004; returned for revision January 31, 2005; accepted February 1, 2005.

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