Articles
IDENTIFICATION AND CHARACTERIZATION OF PRUNASIN HYDROLASES IN SWEET AND BITTER ALMONDS AND THEIR EXPRESSION IN NICOTIANA BENTHAMIANA PLANTS
Article number
1028_12
Pages
83 – 89
Language
English
Abstract
Almond bitterness, caused by high amygdalin content, is the most important trait since bitter kernelled seedlings are usually discarded from breeding programs.
Amygdalin and its precursor prunasin, both cyanogenic glucosides, are degraded by specific enzymes called β-glucosidases.
In order to deep in the genetic control of almond bitterness some works showed differences in the localization of prunasin hydrolases (PH, the β-glucosidase which 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 sweet or bitter taste of their seeds.
RNA was extracted from tegument, nucellus and cotyledon of two sweet (Lauranne and Ramillete) and two bitter (D05-187 and S3067) almonds.
Sequences of twelve positive clones of PH were obtained of all genotypes by RT-PCR and cloning.
These clones were expressed in a heterologous system in Nicotiana benthamiana plants by agroinfiltration.
The activity of this enzyme was detected using the substrate prunasin by the Feigl-Anger method and quantifying the cyanide released.
Furthermore, β-glucosidase activity was detected by Fast Blue BB salt gel.
Ten out of twelve PHs showed activity.
Differences in the sequences of nucleotides and amino acids of PH were found, although these differences were not detected in the activity assays.
Amygdalin and its precursor prunasin, both cyanogenic glucosides, are degraded by specific enzymes called β-glucosidases.
In order to deep in the genetic control of almond bitterness some works showed differences in the localization of prunasin hydrolases (PH, the β-glucosidase which 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 sweet or bitter taste of their seeds.
RNA was extracted from tegument, nucellus and cotyledon of two sweet (Lauranne and Ramillete) and two bitter (D05-187 and S3067) almonds.
Sequences of twelve positive clones of PH were obtained of all genotypes by RT-PCR and cloning.
These clones were expressed in a heterologous system in Nicotiana benthamiana plants by agroinfiltration.
The activity of this enzyme was detected using the substrate prunasin by the Feigl-Anger method and quantifying the cyanide released.
Furthermore, β-glucosidase activity was detected by Fast Blue BB salt gel.
Ten out of twelve PHs showed activity.
Differences in the sequences of nucleotides and amino acids of PH were found, although these differences were not detected in the activity assays.
Authors
J. del Cueto, F. Dicenta, K. Jørgensen, B.L. Møller, R. Sánchez-Pérez
Keywords
Prunus dulcis, prunasin hydrolase, bitterness, β-glucosidases, agroinfiltration
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