Cloning and characterization of two members of the chalcone synthase gene family from walnut

Based on PCR technology two full length chalcone synthase (CHS) cDNAs with about 98 % identity at the nucleic acid level between each other were isolated from leaves of adult walnut tree. The open reading frame sequences coded for two 389-amino acid polypeptides with 99.5 % identity. The two deduced amino acid sequences exhibited the typical CHS consensus in the middle of the sequences and the essential amino acids for CHS (EC 2.3.1.74) activity; in addition, they showed a high degree of identity to the CHS of Pyrus malus (91 %) and Pinus sylvestris (85 %). Perfect repeats were detected in the single intron of one genomic CHS clone isolated. Southern blot analysis indicated that in walnut, CHS is encoded by a small gene family. Sequence analysis of the promoter region of one member of the CHS gene family revealed putative cis-regulatory elements, such as a G-box, H-box and W-box. Accumulation of a 1.5-kb CHS transcript was detected in leaves, buds, liber and bark, whereas no transcripts were detected in wood and medulla. Growth of walnut shoots resulted in a transient increase of leaf and liber CHS transcripts which was much more pronounced in adult than in rejuvenated tree shoots. These data which are correlated with CHS enzyme activity and flavonoid accumulation indicate a tissue-specific, as well as an influence caused by old age on flavonoid biosynthesis, controlled at the transcriptional level.