β-Glucosidase activity in almond seeds

Almond bitterness is the most important trait for breeding programs since bitter-kernelled seedlings are usually discarded. Amygdalin and its precursor prunasin are hydrolyzed by specific enzymes called β-glucosidases. In order to better understand the genetic control of almond bitterness, some studies have shown differences in the location of prunasin hydrolases (PH, the β-glucosidase that degrades prunasin) in sweet and bitter genotypes. The aim of this work was to isolate and characterize different PHs in sweet- and bitter-kernelled almonds to determine whether differences in their genomic or protein sequences are responsible for the sweet or bitter taste of their seeds. RNA was extracted from the tegument, nucellus and cotyledon of one sweet (Lauranne) and two bitter (D05-187 and S3067) almond genotypes throughout fruit ripening. Sequences of nine positive Phs were then obtained from all of the genotypes by RT-PCR and cloning. These clones, from mid ripening stage, were expressed in a heterologous system in tobacco plants by agroinfiltration. The PH activity was detected using the Feigl-Anger method and quantifying the hydrogen cyanide released with prunasin as substrate. Furthermore, β-glucosidase activity was detected by Fast Blue BB salt and Umbelliferyl method. Differences at the sequence level (SNPs) and in the activity assays were detected, although no correlation with bitterness was found.