Bitterness in almonds

Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for beta-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized beta-glucosidase activity, whereas in the bitter cultivar, the beta-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the beta-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.     

Investigation of the microheterogeneity and aglycone specificity-conferring residues of black cherry prunasin hydrolases

In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.     

Pattern of the Cyanide-Potential in Developing Fruits : Implications for Plants Accumulating Cyanogenic Monoglucosides (Phaseolus lunatus) or Cyanogenic Diglucosides in Their Seeds (Linum usitatissimum, Prunus amygdalus)

The absolute cyanide content of developing fruits was determined in Costa Rican wild lima beans (Phaseolus lunatus), oil flax (Linum usitatissimum), and bitter almonds (Prunus amygdalus). The cyanide potential (HCN-p) of the lima bean and the almond fruit began to increase shortly after anthesis and then stopped before fruit maturity. In contrast, the flax inflorescence had a higher HCN-p in absolute terms than the mature flax fruit. At all times of its development the bean fruit contained the monoglucosides linamarin and lotaustralin. The almond and the flax fruits contained, at anthesis, the monoglucosides prunasin, and linamarin and lotaustralin, respectively, while, at maturity, only the corresponding diglucosides amygdalin, and linustatin and neolinustatin, respectively, were present.     

Utilization of Amygdalin during Seedling Development of Prunus serotina

Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside (R)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 [mu]mol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPG:mandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and [beta]-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.     

Development of the Potential for Cyanogenesis in Maturing Black Cherry (Prunus serotina Ehrh.) Fruits

Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase I (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase II, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases II (29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages.     

Evolutionary analysis of S-RNase genes from Rosaceae species

Eight new cDNA sequences for S-RNases were cloned and analysed from almond (Prunus dulcis) cultivars of European origin, and compared to published sequences from other Rosaceae species. Insertions/deletions of 10-20 amino acid residues were detected in the RC4 and C5 domains of S-RNases from almond and sweet cherry. The S-RNases of the Prunus species and those of the genera Malus and Pyrus formed two distinct groups on phylogenetic analysis. Nucleotide substitutions were analysed in the S-RNase genes of these species. The S-genes of almond and sweet cherry have a lower Ka/Ks value than those of apple, pear and wild apple do. The fact that there is no fixed difference between the S-RNase genes of almond and sweet cherry, or between apple and pear, suggests that nucleotide substitutions only introduce transient polymorphism into the two groups, and rarely became fixed and contribute to divergence. Through the comparative study of 17 S-RNase genes from the genus Prunus and 18 from the genera Malus and Pyrus, some fixed nucleotide differences between the two groups were identified. These differences do not appear to be the result of selection for adaptive mutations, since the number of replacement substitutions is not significantly greater than the number of synonymous substitutions. S-RNase genes of almond and sweet cherry, and of apple and pear, showed little heterogeneity in nucleotide substitution rates. However, heterogeneity was observed between the two groups of S-alleles, with the Prunus alleles exhibiting a lower rate of non-synonymous substitutions than alleles from Malus and Pyrus. The evolutionary relationships between these species are discussed.     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the beta-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components.     

In silico authentication of amygdalin as a potent anticancer compound in the bitter kernels of family Rosaceae

Amygdalin a naturally occurring compound, predominantly in the bitter kernels of apricot, almond, apple and other members of Rosaceae family. Though, amygdalin is used as an alternative therapy to treat various types of cancer but its role in cancer pathways has rarely been explored yet. Therefore, present study was intended with the aim to investigate the alleged anti-cancerous effects of amygdalin specifically on PI3K–AKT–mTOR and Ras pathways of cancer in human body. Computational modelling and simulation techniques were used to assess the effect of amygdalin on PI3K-AKT-mTOR and Ras pathways using different level of dosage. It was observed that amygdalin had direct and substantial contribution to regulate PI3K-mTOR activities on threshold levels while the other caner pathways were effected indirectly. Consequently, amygdalin is a down-regulator of a cancer within a specified amount and contribute considerably to reduce various types of cancer in human. Furthermore, in-vitro and in-vivo analyses of amygdalin could be of helpful to authenticate its pharmacological effects.     

Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds

Plum (Prunus domestica) seeds, which contain the cyanogenic diglucoside (R)-amygdalin and lesser amounts of the corresponding monoglucoside (R)-prunasin, release the respiratory toxin HCN upon tissue disruption. Amygdalin hydrolase (AH) and prunasin hydrolase (PH), two specific [beta]-glucosidases responsible for hydrolysis of these glucosides, were purified to near homogeneity by concanavalin A-Sepharose 4B and carboxymethyl-cellulose chromatography. Both proteins appear as polypeptides with molecular masses of 60 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but they exhibit different isoelectric points (PH, 5.6-6.0; AH, 7.8-8.2). AH and PH were localized within mature plum seeds by tissue printing, histochemistry, and silver-enhanced immunogold labeling. As was previously shown in black cherry (Prunus serotina) seeds (E.Swain, C.P. Li, J.E. Poulton [1992] Plant Physiol 100: 291-300), AH and PH are restricted to protein bodies of specific procambial cells and are absent from the cotyledonary parenchyma, bundle sheath, and endosperm cells. In contrast, the cyanogenic glycosides in both plum and black cherry seeds, which were detected by tissue printing, occur solely in the cotyledonary parenchyma and are absent from the procambium and endosperm. It is concluded that tissue level compartmentation prevents large-scale cyanoglycoside hydrolysis in intact Prunus seeds.     

Differences in body size and egg loads of Rhagoletis indifferens (Diptera: Tephritidae) from introduced and native cherries

The western cherry fruit fly, Rhagoletis indifferens Curran, infests introduced, domesticated sweet [Prunus avium (L.) L.], and tart cherries (Prunus cerasus L.) as well as native bitter cherry, Prunus emarginata (Douglas) Eaton. Bitter cherries are smaller than sweet and tart cherries and this could affect various life history traits of flies. The objectives of the current study were to determine 1) if body size and egg loads of flies infesting sweet, tart, and bitter cherries differ from one another; and 2) if any observed body size differences are genetically based or caused by the host fruit environment. Pupae and adults of both sexes reared from larval-infested sweet and tart cherries collected in Washington and Montana were larger than those reared from bitter cherries. In addition, flies of both sexes caught on traps in sweet and tart cherry trees were larger than those caught in bitter cherry trees and females trapped from sweet and tart cherry trees had 54.0-98.8% more eggs. The progeny of flies from naturally-infested sweet and bitter cherries reared for one generation in the laboratory on sweet cherry did not differ in size. The same also was true for progeny of sweet and bitter cherry flies reared in the field on bitter cherry. The results suggest that the larger body sizes of flies from sweet and tart cherries than bitter cherries in the field are caused by host fruit and not genetic factors.     

Probing behavior of bird cherry-oat aphid Rhopalosiphum padi (L.) on native bird cherry Prunus padus L. and alien invasive black cherry Prunus serotina Erhr. in Europe and the role of cyanogenic glycosides

The probing behavior of bird cherry-oat aphid Rhopalosiphum padi was studied on its natural winter host in Europe, the bird cherry Prunus padus, and on the invasive black cherry Prunus serotina, on which spring generations of R. padi do not survive. The EPG-recorded behavior of R. padi on bird cherry and black cherry showed differences in crucial aspects of probing and feeding. The period of the pre-phloem penetration was twice as long and rarely interrupted in aphids on bird cherry as opposed to aphids on black cherry. On black cherry, there was a considerable delay between finding and accepting the phloem. Aphids that had sampled phloem sap either refused to ingest it or the ingestion periods were very short. Amygdalin and prunasin (cyanogenic glycosides present in leaves of Prunus) seriously impeded ingestion activities when applied in pure sucrose diet. The role of amygdalin and prunasin in winter host plant selection and host alternation in R. padi is discussed.     

Molecular markers for kernel bitterness in almond

Upon crushing, amygdalin present in bitter almonds is hydrolysed to benzaldehyde, which gives a bitter flavour, and to cyanide, which is toxic. Bitterness is attributable to the recessive allele of the Sweet kernel (Sk/sk) gene and is selected against in breeding programmes. Almond has a long intergeneration period due to its long juvenile phase, so breeders must wait 3 or 4 years to evaluate fruit traits in the field. For this reason, it is important to develop molecular markers to distinguish between sweet and bitter genotypes. The Sk gene is known to map to linkage group five (G5) of the almond genome, but its function is still undefined. Candidate genes involved in the amygdalin pathway have been mapped, but none of them were located to G5. We have saturated G5 with additional Simple Sequence Repeats (SSRs) using the progeny from the cross “R1000” × “Desmayo Largueta” and found six SSRs (UDA-045, EPDCU2584, CPDCT028, BPPCT037, PceGA025, and CPDCT016) closely linked to the Sk locus. The genotypes of four of these SSRs flanking the Sk locus, in a number of parents and a few seedlings of the CEBAS-CSIC almond breeding programme, allowed us to estimate the haplotypes of the parents, identifying the marker alleles adequate for an early and highly efficient selection against bitter genotypes. This analysis has established the usefulness of SSRs for screening populations of fruit trees such as almond by an easy, polymerase chain reaction-based method.     

Catalytic degradation of amygdalin by extracellular enzymes from Aspergillus niger

Amygdalin is a controversial anti-tumor natural product that has been used as an alternative cancer drug for many years. The anti-tumor mechanism and metabolism of amygdalin have been the focus of many studies. However, previous studies by our group demonstrated that amygdalin itself has no anti-tumor activity, but rather the active ingredients were determined to be amygdalin degradation products. To screen novel drugs with anti-tumor activity, the extracellular enzymes from Aspergillus niger were used to degrade amygdalin. Within 4 h of the catalytic reaction at 37°, amygdalin was rapidly degraded into four products. The products were then extracted and purified by column chromatography. By comparing the HPLC chromatograms, ¹H NMR, ¹³C NMR and MS data, the products were identified as mandelonitrile, prunasin, benzaldehyde and phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile (PTMT), a novel hydroxyl derivative of prunasin. Furthermore, pharmacology studies of these compounds demonstrated that 10 mg/kg of PTMT significantly suppressed the growth of S-18 tumor cells within 11 days in a concentration-dependent manner.     

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.     

Microsatellites isolated in almond from an AC‐repeat enriched library

We have isolated 44 SSRs from an AC‐enriched genomic library from almond (Prunus amygdalus Batsch.). Twenty SSRs were screened for their polymorphism in 16 cultivars and for their transportability in seven different Prunus species (peach, nectarine, apricot, European plum, Japanese plum, sweet cherry, sour cherry) and in apple. The expected heterozygosity ranged from 0.62 to 0.89. About 30% of primers gave successful amplification in seven different Prunus species; in two cases amplifications were obtained also in apple.     

Simple screening procedure for microorganisms to degrade amygdalin

Summary Amygdalin is a cyanogenic glycoside occurring among others in almonds and bitter apricot seeds. Utilization of seeds for human or animal nutrition requires adequate detoxification. The present method for screening microbial cultures for their ability to degrade amygdalin is a convenient micro-titre version based on the picrate reaction principle.     

Molecular cloning of the cDNA coding for the (R)-(+)-mandelonitrile lyase of Prunus amygdalus: temporal and spatial expression patterns in flowers and mature seeds

A gene highly expressed in the floral organs of almond (Prunus amygdalus Batsch), and coding for the cyanogenic enzyme (R)-(+)-mandelonitrile lyase (EC 4.1.2.10), has been identified and the full-length cDNA sequenced. The temporal expression pattern in maturing seeds and during floral development was analyzed by RNA blot, and the highest mRNA levels were detected in floral tissues. The spatial mRNA accumulation pattern in almond flower buds was also analyzed by in-situ hybridization. The mRNA levels were compared during seed maturation and floral development in fruit and floral samples from cultivars classified as homozygous or heterozygous for the sweet-almond trait or homozygous for the bitter trait. No correlation was found between these characteristics and levels of mandelonitrile lyase mRNA, suggesting that the presence of this protein is not the limiting factor in the production of hydrogen cyanide. Received: 3 December 1997 / Accepted: 18 April 1998     

Tissue and cellular localization of individual β‐glycosidases using a substrate‐specific sugar reducing assay

Traditional methods to localize β‐glycosidase activity in tissue sections have been based on incubation with the general substrate 6‐bromo‐2‐naphthyl‐β‐d ‐glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6‐bromo‐2‐naphthyl‐β‐d ‐glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different β‐glycosidases present in the tissue. To be able to monitor the occurrence of individual β‐glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any β‐glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5‐triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two β‐glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.