Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

Effects of the overexpression of a soybean cytosolic glutamine synthetase gene (GS15) linked to organ-specific promoters on growth and nitrogen accumulation of pea plants supplied with ammonium.

A soybean cytosolic glutamine synthetase gene (GS15) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)), or a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies (Lines 1, 7, 8 and 9) and four lines with two copies each of GS15 (Lines 2, 4, 6 and 11) were compared to the wild-type (WT) parental line for levels of cytosolic glutamine synthetase (GS1), glutamine synthetase (GS) activity, N accumulation, N derived form the atmosphere (NDFA), and biomass of plants grown on 0.0, 0.1, 1.0 or 10.0 mM NH(4)(+). Enhanced levels of GS1 were detected in leaves of one of the two lines transformed with the 35S-GS15 construct, and all three lines containing the rolD-GS15 construct. All three lines containing the LBC(3)-GS15 construct had increased levels of GS1 in nodules. Despite the increased levels of GS1 in many transformants, only the roots of lines containing the rolD-GS15 construct consistently demonstrated enhanced levels of GS activity (up to 12-fold). Positive responses in plant N content, NDFA, and biomass were rare, but increases in plant biomass and N content of up to 17% and 54%, respectively, occurred in some of the rolD-GS15 lines at certain levels of ammonium. In general, GS15 copy number did not seem to differentially affect phenotype of the transformants, and transformants respond to ammonium concentrations in similar patterns to that previously observed with nitrate. Despite the fact that the rolD-GS15 transformants consistently resulted in increased GS activity in roots and resulted in some occurrences of increases in biomass and plant N content, the lack of consistent positive growth effect across all transformants indicates that the generalized overexpression of GS1 in tissues holds little potential for positive growth responses in pea.     

Kinetic properties of microsomal UDP-glucuronyltransferase. Regulation by metal ions

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V_max. The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.     

Structural characterization of cholylcoenzyme A:glycine-taurine N-acyltransferase and a covalent substrate intermediate

Bile acid-CoA:glycine-taurine N-acyltransferase was found to catalyze a reaction in the absence of glycine or taurine in which the substrate cholyl-CoA is cleaved with the release of CoA and the formation of a covalently bound enzyme-cholate intermediate. This unstable intermediate was trapped by a rapid mixing and denaturation procedure. The denatured protein was digested with trypsin and the cholate-labeled tryptic peptide was isolated. This cholate-peptide is considered to originate from the active site region of the enzyme based on the following criteria: cholyl-CoA does not react with any of the 20 common amino acids, the hydrolysis of cholyl-CoA is known to occur on the enzyme, the lack of reaction of the enzyme with just cholate, and the fact that labeling is extensive even at low (substrate level) concentrations of cholyl-CoA. The isolated cholate-peptide was submitted to amino acid analysis. It contained 32 amino acid residues and was devoid of cysteine, methionine, and tyrosine. Amino acid analysis of the N-acyltransferase was conducted. The enzyme was also shown to possess a blocked N terminus.