title: Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions.
abstract: Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.
authors.affiliation: Department of Agricultural Sciences, University of Bristol, UK.
authors.forename: R N
authors.initials: RN
authors.lastname: Waterhouse
authors.forename: A J
authors.initials: AJ
authors.lastname: Smyth
authors.forename: A
authors.initials: A
authors.lastname: Massonneau
authors.forename: I M
authors.initials: IM
authors.lastname: Prosser
authors.forename: D T
authors.initials: DT
authors.lastname: Clarkson
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001216:Asparagine / Q000096:biosynthesis*; D001217:Aspartate-Ammonia Ligase / Q000235:genetics*; D002940:Circadian Rhythm; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D018517:Plant Roots / Q000378:metabolism; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D018076:DNA, Complementary; D009587:Nitrogen Isotopes; D001216:Asparagine; D001217:Aspartate-Ammonia Ligase