Aiuga reptans is a frost-hardy, perennial labiate that is known for its high content of raffinose family oligosaccharide(s) (RFO). Seasonal variations in soluble nonstructural carbohydrate levels in above-ground parts of Aiuga showed that the RFO were by far the most predominant components throughout the whole year. RFO were lowest in summer (75 mg/g fresh weight) and highest in fall/ winter (200 mg/g fresh weight), whereas sucrose and starch were only minor components. Cold treatment (14 d at 10/3"C, day/ night) of plants that were precultivated under warm conditions (25'C) lowered the temperature optimum of net photosynthesis from 16' to 8"C, decreased the maximum rate, and increased the total nonstructural carbohydrate content of leaves by a fador of about 10, mainly because of an increase of RFO. The degree of polymerization of the R F O increased sequentially up to at least 15. A novel, galactinol-independent galactosyltransferase enzyme was found, forming from two molecules of RFO, the next higher and lower degree of polymerization of RFO. The enzyme had a pH optimum of 4.5 to 5.0 and may be responsible for RFO chain elongation. RFO were the main carbohydrates translocated in the phloem, with stachyose being by far the most dominant form. Studies of carbon balance during leaf development revealed a transition point between import and export at approximately 25% maximal leaf area. RFO synthesis could be detected even before the commencement of export, suggesting the existence of a nonphloem-linked RFO pool even in very young leaves. Taken together, it seems that Ajuga leaves contain two pools of RFO metabolism, a pronounced long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem.RFO (a-1,6-galactosyln-Suc; 1 5 n < approximately 7) are the most widely occuning oligosaccharides in the plant kingdom. RFO synthesis proceeds by sequential action of a series of a-galactosyltransferases (Kandler and Hopf, 1984; Dey, been found (Hérissey et al., 1954) that are thought to be synthesized sequentially by specific galactosyltransferases using galactinol as the Gal donor analogously to the synthesis of raffinose and stachyose. RFO catabolism proceeds by sequential exo-action of a-Gal(s).Almost a11 biochemical and physiological studies conceming RFO metabolism have been performed on leaves of cucurbits and seeds of legumes. The specialization on these non-frost-hardy plants may lead to a bias with regard to the roles of the RFO in plant metabolism. The main functions of the RFO are still believed to be carbon transport in leaves and desiccation tolerance and carbon storage in seeds. Less attention has been given to equally important functions of the RFO such as cold tolerance (e.g. in conifer needles, Hinesley et al., 1992; Wiemken and Ineichen, 1993) and carbon storage (eg. in Stachys tubers, Keller and Matile, 1985) in vegetative plant parts. To increase our knowledge of RFO metabolism in these less well-studied areas, we needed a suitable experimental pl...

The inhibitor protein (IP) that inactivates spinach leaf N ADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GFI4¢o, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14¢o.

Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: to > Z > a) >>> ~, ¥.