Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

Abstract

We previously reported on Vicia narbonensis seeds with largely decreased alpha- 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 antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]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, phospho enolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, 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.

Enhanced plant tolerance to iron starvation by functional substitution of chloroplast ferredoxin with a bacterial flavodoxin

“…The collected filtrate was used for ion chromatography coupled to mass spectrometry analysis according to published procedures (35). Soluble amino acids were determined as described (36). Numerical analysis was carried out by using Empower Pro software (Waters, Milford, MA).…”

Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis

“…After evaporating the supernatant to dryness, the amino acids and standards were derivatized as described previously (Rolletschek et al, 2002) using AQC (Watrex). The AQC amino acids were measured using a Dionex P680-HPLC system with an UV170U detector (Dionex) and the column system consisting of CC8/4 ND 100-5 C18-ec and Nucleodur 100-5 C18-ec (Macherey-Nagel).…”

RNAi-Mediated Tocopherol Deficiency Impairs Photoassimilate Export in Transgenic Potato Plants

“…Extracts were evaporated to dryness, redissolved in purest H 2 O, and separated on a reversed-phase HPLC system (Waters Associates, Milford, MA) after derivatization using the AccQ-Tag method as described (Rolletschek et al, 2002). Amino acids were identified by cochromatography with authentic standards and quantified by comparison with internal standards.…”