MOLECULAR-CLONING AND CHARACTERIZATION OF ASPARAGINE SYNTHETASE FROM LOTUS-JAPONICUS - DYNAMICS OF ASPARAGINE SYNTHESIS IN N-SUFFICIENT CONDITIONS

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthe tases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequen ces indicated 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 transcri pt were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expressio n are different from those seen in pea. AS gene expression was monitor ed throughout a 16 h light/8 h dark day, under nitrate-sufficient cond itions. Neither transcript showed the dark-enhanced accumulation patte rns previously reported for other plant AS genes. To evaluate AS activ ity, the molecular dynamics of asparagine synthesis were examined in v ivo using N-15-ammonium labelling. A constant rate of asparagine synth esis in the roots was observed. Asparagine was the most predominant am ino-component of the xylem sap and became labelled at a slightly slowe r rate than the asparagine in the roots, indicating that most root asp aragine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the N-15-la belling data suggest that light regulation of AS gene expression is no t a factor controlling N-assimilation in L. japonicus roots during sta ble growth in N-sufficient conditions.