ORIGINAL ARTICLE Hardy Rolletschek ® Mohammad-Reza Hajirezaei Ulrich Wobus ® Hans Weber Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake Received: 13 June 2001 / Accepted: 25 October 2001 / Published online: 23 January 2002 Ó Springer-Verlag 2002 Abstract We previously reported on Vicia narbonensis seeds with largely decreased a-D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33–43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of anti- sense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in par- ticular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legu- min to vicilin. Isolated cotyledons partitioned less [ 14 C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon parti- tioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble com- pounds were increased but osmolality remained un- changed, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx. Keywords Amino acid uptake ® Carbon partitioning ® Seed (development, water relations) ® Starch biosynthesis ® Storage protein synthesis ® Vicia (carbon partitioning) Abbreviations AGP: ADP-glucose pyrophosphorylase ® DAP: days after pollination ® G-3-P: glyceraldehyde-3- phosphate ® Glc-6-P: glucose-6-phosphate ® Fru-1,6-P 2 : fructose-1,6-bisphosphate ® PEP: phosphoenolpyruvate ® 3-PGA: 3-phosphoglyceric acid Introduction Genetic modification of metabolic pathways is a pow- erful way to analyze seed development. We recently created transgenic Vicia narbonensis L. lines expressing the small-subunit gene of ADP-glucose pyrophosphor- ylase (AGP) in antisense orientation under the control of the legumin B4 storage-protein promoter, which confers expression in the storage parenchyma of maturing cot- yledons. The plastidial enzyme AGP catalyzes the conversion of glucose-1-phosphate and ATP to pyro- phosphate and ADP-glucose, which is the substrate for starch synthase. We were able to show that transgenic cotyledons contained moderately decreased amounts of starch. Transgenic seeds accumulated dry weight during a longer time period and, therefore, had a prolonged seed-filling period (Weber et al. 2000). The protein content was higher but it is unclear whether this was caused by altered carbon partitioning, nitrogen uptake or to the longer seed-filling period. The primary aim of this paper was therefore to establish the relationship between these processes. Legume seeds accumulate starch and storage pro- teins. The latter consist of two major classes, globulins and albumins: 11S legumins and 7S vicilins account for Planta (2002) 214: 954–964 DOI 10.1007/s00425-001-0710-4 H. Rolletschek ® M.-R. Hajirezaei ® U. Wobus ® H. Weber (&) Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung (IPK), 06466 Gatersleben, Germany E-mail: weber@ipk-gatersleben.de Fax: +49-39482-5138