Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis)

Abstract

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a β(1→6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana. Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.

Glycoside‐specific glycosyltransferases catalyze regio‐selective sequential glucosylations for a sesame lignan, sesaminol triglucoside

“…DDBJ/EMBL/GenBank accession numbers for UGT94AB2, UGT94AA2, and UGT94AG1 are LC484012, LC484014, and LC484013, respectively. The regio-selectivity of GGTs used in the analysis was described in the following studies: Brugliera et al (1994), Kroon et al (1994), Frydman et al (2004 (2014,2016), Di et al (2015), Ohgami et al (2015), Casas et al (2016), and Thodberg et al (2018).…”

Transposons played a major role in the diversification between the closely related almond (Prunus dulcis) and peach (P. persica) genomes: Results from the almond genome sequence

“…Our results suggest that TEs could be responsible for some of the genomic changes at the origin of the agronomic traits that distinguish peach from almond, such as mesocarp development and bitterness of the kernel. For one of them, sweet vs. bitter kernel, essential for the domestication of the almond, we show here that the sweet almond phenotype correlates with TE insertions surrounding gene CYP71AN24, involved in the synthesis of one of the key enzymes of the amygdalin pathway (cytochrome P450), and that could be responsible for the lower expression of this gene in the seed tegument of sweet almonds (Thodberg et al 2018).…”

“…It has been recently shown that the sweet almond phenotype is due to the reduced expression of the genes encoding two cytochrome P450 enzymes catalyzing the first steps of the amygdalin biosynthesis in sweet almond varieties as compared with bitter almond varieties (Thodberg et al 2018). It has also been shown that this reduced expression is not related with differences in the gene sequence, which points to a difference in the regulation of the expression of those genes (Thodberg et al 2018). We have compared these two almond genes with their homologs in peach, sweet cherry and P. mume and found that they are highly conserved (not shown).…”

Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudian

“…5). Interestingly, PdUGT94AF1 and PdUGT94AF2 derived from Prunus dulcis involved in the formation 1,6-β-D-glucosidic linkage of Prunasin [24] were clustered in group A but had a long genetic distance between them and SrUGTs of this group, implying that the SrUGTs in this group may have no related specificity. Due to the lack of other glycosyltransferases capable of forming 1,6-glucosidic bonds, this tree cannot predict the glycosyltransferases involved in the synthesis of steviol glycosides containing 1,6-glucosidic bonds (such as Reb L).…”