Bitterness in Almonds1[C][OA] · mation in plants has its origin in antiquity. In ancient Egypt, traitorous priests in Memphis and Thebes were poisoned to death with pits of peaches

Bitterness in Almonds1[C][OA]

Raquel Sanchez-Perez, Kirsten Jørgensen, Carl Erik Olsen, Federico Dicenta, and Birger Lindberg Møller*

Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology(R.S.-P, K.J., B.L.M.), and Chemistry Department (C.E.O.), Faculty of Life Sciences, University of Copenhagen,DK–1871 Frederiksberg C, Copenhagen, Denmark; and Departamento de Mejora Vegetal, Centrode Edafologıa y Biologıa Aplicada del Segura-Consejo Superior de Investigaciones Cientıficas, E–30100Murcia, Spain (F.D.)

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

The knowledge about hydrogen cyanide (HCN) for-mation in plants has its origin in antiquity. In ancientEgypt, traitorous priests in Memphis and Thebes werepoisoned to death with pits of peaches (Davis, 1991).The first known detection of HCN liberated fromdamaged plant tissue was made in 1802 by the phar-macist Bohm in Berlin upon distillation of bitter al-monds (Lechtenberg and Nahrstedt, 1999). In 1830,Robiquet and Boutron-Chalard discovered the struc-ture of the HCN-liberating compound in bitter almonds(Lechtenberg and Nahrstedt, 1999). Because the com-pound was isolated from Prunus amygdalus (synonymPrunus dulcis), it was named amygdalin. Amygdalinhas subsequently been found widespread in seeds ofother members of the Rosaceae like in apples (Malusspp.), peaches (Prunus persica), apricots (Prunus arme-

niaca), black cherries (Prunus serotina), and plums(Prunus spp.; McCarty et al., 1952; Conn, 1980; Frehneret al., 1990; Møller and Seigler, 1991; Swain et al., 1992;Poulton and Li, 1994; Arrazola, 2002; Dicenta et al.,2002). The diglucoside amygdalin was the first memberto be isolated of a new class of natural products nowknown as cyanogenic glucosides. Cyanogenic gluco-sides are present in more than 2,500 different plantspecies, including many important crop plants (Seiglerand Brinker, 1993; Bak et al., 2006). Upon disruption ofplant tissue containing cyanogenic glucosides, these aretypically hydrolyzed by b-glucosidases with concomi-tant release of Glc, an aldehyde or ketone, and HCN.This two-component system, of which each of the sep-arate components is chemically inert, provides plantswith an immediate chemical defense against attackingherbivores and pathogens (Conn, 1969; Nahrstedt, 1985;Jones, 1988; Morant et al., 2003; Nielsen et al., 2006;Zagrobelny et al., 2004, 2007a, 2007b). In addition totheir possible defense function, accumulation of cyano-genic glucosides in certain angiosperm seeds may pro-vide a storage deposit of reduced nitrogen and sugarfor the developing seedlings (Lieberei et al., 1985;Selmar et al., 1988, 1990; Swain et al., 1992).

Caius Plinius Secundus (better known as Pliny theElder) stated in his 37-volume encyclopedia entitledNaturalis Historia, which was completed shortly afterhis death in 79 AD that the Romans were proud of know-ing how to remove bitterness from almond kernels(Pliny the Elder, 77; Alvarez, 1798). Nevertheless, sweetkernel in almond remains the prime breeding target for

1 This work was supported by the Danish National ResearchFoundation (to the Center for Molecular Plant Physiology) and theSpanish Ministry of Education and Science, including a postdoctoralfellowship (to R.S.-P.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Birger Lindberg Møller ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.112979

1040 Plant Physiology, March 2008, Vol. 146, pp. 1040–1052, www.plantphysiol.org � 2008 American Society of Plant Biologists

almond breeders and growers. Sweet kernel in almondhas been shown to be a monogenic trait and the bitterkernel trait to be recessive (Heppner, 1923, 1926; Dicentaand Garcıa, 1993; Dicenta et al., 2007). The gene confer-ring sweetness (Sweet kernel [Sk] gene) belongs to linkagegroup five (Joobeur et al., 1998; Bliss et al., 2002;Sanchez-Perez et al., 2007) but the precise localizationand function of this maternally inherited gene remainsunknown.

Previous studies in almonds have shown that pru-nasin is transformed into amygdalin during fruitripening (Frehner et al., 1990). Prunasin is present inroots, leaves, and kernels of sweet, slightly bitter andbitter varieties (Dicenta et al., 2002). Tracer experi-ments have demonstrated that prunasin is synthesizedfrom Phe (Mentzer and Favre-Bonvin, 1961). Sorghum(Sorghum bicolor) contains the Tyr-derived cyanogenicglucoside dhurrin (Akazawa et al., 1960; Møller andPoulton, 1993). Dhurrin biosynthesis is catalyzed bytwo multifunctional membrane-bound cytochromeP450 (Cyt P450) enzymes CYP79A1 and CYP71E1(Sibbesen et al., 1994, 1995; Bak et al., 1998, 2000).CYP79A1 catalyzes the conversion of Tyr into Z-p-hydroxyphenylacetaldoxime (Sibbesen et al., 1995)and CYP71E1 catalyzes the conversion of the Z-p-hydroxyphenylacetaldoxime into p-hydroxymandelo-nitrile (Kahn et al., 1997; Bak et al., 1998). Conversionof the labile cyanohydrin into dhurrin is catalyzed by asoluble UDP-Glc (UDPG)-glucosyltransferase UGT85B1(Jones et al., 1999; Hansen et al., 2003). The entire path-way for dhurrin synthesis has been transferred fromsorghum to Arabidopsis (Arabidopsis thaliana) usinggenetic engineering (Tattersall et al., 2001; Kristensenet al., 2005). Prunasin biosynthesis is thought to followthe same biosynthetic scheme as dhurrin biosynthesisbut no enzymes or genes involved have been identified(Møller and Seigler, 1991). Prunasin is converted intothe diglucoside amygdalin by means of an additionalUDPG-glucosyltransferase (Fig. 1). In contrast to the

situation with the biosynthetic enzymes in almond,the enzymes and genes involved in amygdalin degra-dation have been identified and characterized. Thefirst step in amygdalin degradation is mediated by theb-glucosidase amygdalin hydrolase (EC 3.2.1.117) andresults in the formation of prunasin and concomitantrelease of Glc. Prunasin is subsequently hydrolyzed byanother b-glucosidase named prunasin hydrolase (EC3.2.1.21) to form mandelonitrile and Glc. Mandeloni-trile is finally converted into benzaldehyde and HCNby the action of mandelonitrile lyase (EC 4.1.2.10;Swain and Poulton, 1994a). This conversion may alsoproceed nonenzymatically at neutral or alkaline pH.HCN is an inhibitor of cell respiration and is detoxifiedby the action of the enzyme b-cyano-Ala synthase,which converts HCN and Cys into b-cyano-Ala (Flosset al., 1965). By the action of nitrilases, b-cyano-Ala isconverted into Asn and Asp (Swain and Poulton,1994b; Piotrowski et al., 2001; Piotrowski and Volmer,2006; Jenrich et al., 2007; Kriechbaumer et al., 2007).

In this study we have investigated prunasin andamygdalin synthesis and turnover in sweet and bitteralmond genotypes by direct measurements of the en-zyme activities and their tissue and cellular locations.The results point to a difference in b-glucosidase activ-ity in the inner epidermis of the tegument as the maindeterminant of whether a variety is sweet or bitter.

RESULTS

Fruit Development and Ripening

The synthesis, accumulation, and degradation of thecyanogenic glucosides prunasin and amygdalin werestudied during the entire growth season from treeflowering to full ripening of the kernel using the fourgenotypes ‘Ramillete’ (SkSk, sweet), ‘Marcona’ (Sksk,sweet), ‘Garrigues’ (Sksk, slightly bitter), and ‘S3067’(sksk, bitter). Almond trees flower in the month of

Figure 1. The metabolic pathways for synthesis and catabolism of the cyanogenic glucosides prunasin and amygdalin inalmonds. Biosynthetic enzymes (black lines) are: CYP79 and CYP71, Cyt P450 monooxygenases; GT1, UDPG-mandelonitrileglucosyltransferase; and GT2, UDPG-prunasin glucosyltransferase. Catabolic enzymes (dashed lines) are: AH, Amygdalinhydrolase; PH, prunasin hydrolase; MDL1, mandelonitrile lyase; and ADGH*, amygdalin diglucosidase (putative).

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February in southern Spain. Fruit development ischaracterized by an increased size of the cotyledon atthe expense of a diminishing nucellus and endosperm(Fig. 2). The development of the different tissues of thefruit in the four genotypes showed sequential devia-tions as has previously been reported in other almondcultivars (Serafimov, 1981). Accordingly, developmentand ripening of the different fruit tissues proceeded ina slightly shifted manner from one genotype to theother. Until April, the maternally derived tissues asexocarp, mesocarp, endocarp, and tegument surroundand protect a liquefied glassy-looking cotyledon. Theendocarp is green and quite soft rendering the fruitseasy to open. In May, the endosperm and a smallgrowing embryo with its characteristic whitish coty-ledons are visible in all genotypes and the size of thenucellus has decreased. The endocarp has becomedifficult to open and is turning brown with a woodyappearance. Finally, at the end of the ripening season,the cotyledons occupy the entire space inside thetegument. The endocarp is hard and the mesocarptogether with the exocarp is beginning to dry eventu-ally exposing the endocarp.

Cyanogenic Glucoside Levels from Flowering to

Fruit Ripening

The levels of prunasin and amygdalin during theentire growth season from tree flowering to fruitripening was monitored by liquid chromatography-mass spectrometry (LC-MS) analyses in the four geno-types ‘Ramillete’ (SkSk, sweet), ‘Marcona’ (Sksk,sweet), ‘Garrigues’ (Sksk, slightly bitter), and ‘S3067’(sksk, bitter; Fig. 3). In the bitter genotype, prunasinwas detected in leaf laminae, petioles, fruit tegument,and nucellus plus endosperm. In leaf laminae andpetioles, the prunasin content peaked in April. Pruna-sin content in tegument from the bitter genotype wasthe highest found in the fruit tissues. Its increase wasfrom March to June. In the three other genotypes,prunasin was present in much lower amounts in leaflaminae and stems and was not detectable in any of thefruit tissues analyzed. No prunasin was detected infruit exocarp, mesocarp, and endocarp of the fourgenotypes (data not shown).

All analyses of teguments from the four genotypesshowed the absence of amygdalin in this fruit tissue.Amygdalin was detectable in the nucellus and endo-

Figure 2. A, The effect of the growth period on the size (width in millimeters) of fruits, seeds, and cotyledons of four genotypes ofalmonds with respect to bitterness: ‘Ramillete’, (SkSk, sweet; ¤), ‘Marcona’ (Sksk, sweet; n), ‘Garrigues’ (Sksk, slightly bitter; :),and ‘S3067’ (sksk, bitter; 3). Measurements were made every second week and initiated March 14, 2007 and ended August 15,2007. Marcona ripened last and therefore this genotype was the only one analyzed on August 15. B, Fruit anatomy of the fouralmond genotypes during the growth period: In March, exocarp (3), green mesocarp (m), a soft endocarp (n), tegument (t), andthe nucellus (u) are visible. In April, the formation of cotyledons (c) is apparent in the earliest varieties. In May, cotyledons (c) andendosperm (n) are being formed at the expense of the nucellus (u). In August, at the end of the ripening season, the cotyledons (c)occupy the entire seed; the endocarp had fully hardened and fragmented during opening. This is the reason the fruits shownappear smaller. [See online article for color version of this figure.]

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1042 Plant Physiol. Vol. 146, 2008

sperm from the bitter genotype. Concomitant withthe decrease in prunasin content in the nucellus andendosperm of the bitter genotype, the amygdalin con-tent in the cotyledons began to increase to reach a finalconcentration of 9 mmol/100 mg fresh weight. In theslightly bitter genotype ‘Garrigues’ and in the sweetheterozygous genotype ‘Marcona’, amygdalin was de-tectable, but at much lower concentrations of 0.03 and0.007 mmol/100 mg fresh weight, respectively. In thesweet homozygous genotype ‘Ramillete’ no amygda-lin was detectable.

Girdling Experiments and Collection of Exudates FormPeduncle Stubs

To investigate whether prunasin or amygdalin wassynthesized in the shoot apex and transported to other

parts of the almond tree and whether transport to thedeveloping almond fruit from other parts of the al-mond tree were occurring, a series of girdling exper-iments were performed in which the epidermis andcambium cell layers, including the phloem, were re-moved to prevent transport across the site of incision.Prior to the girdling, analyses of stems and pedunclesfrom ‘Ramillete’ (SkSk, sweet) and ‘S3067’ (sksk, bitter)showed the presence of prunasin and minute amountsof amygdalin (Fig. 4). The prunasin level was highestin the stem where it reached 2 mmol/100 mg freshtissue in the bitter genotype ‘S3067’ and 0.3 mmol/100mg fresh tissue in the sweet genotype ‘Ramillete’. Thecorresponding values in peduncles were 0.3 and 0.05mmols/100 mg tissue, respectively. The absolute pru-nasin levels in both tissues varied considerably fromone experiment to the other. Amygdalin levels never

Figure 3. Prunasin (solid lines) andamygdalin (bold dashed lines)levels in different parts of the al-mond tree and in different tissues ofthe fruit in the four different geno-types: ‘Ramillete’, (SkSk, sweet;¤), ‘Marcona’ (Sksk, sweet; n),‘Garrigues’ (Sksk, slightly bitter;:), and ‘S3067’ (sksk, bitter; 3).Measurements were made everysecond week and the specific datesare indicated in Figure 2. All fourgenotypes were analyzed and inthe panels where no traces foramygdalin are shown, the levelswere so low that the graph wouldbe superimposed with the x axis.[See online article for color versionof this figure.]

Figure 4. Girdling experiments carried out in April and May with stems beneath the first-year shoot and with peduncles of thegenotypes ‘Ramillete’ (SkSk, sweet; white columns) and ‘S3067’ (sksk, bitter; dashed columns) showing the prunasin andamygdalin content in segments above and below the incision zone. The experimental setup for the girdling of stems andpeduncles is shown in the two right panels. [See online article for color version of this figure.]



exceeded 0.005 mmol/100 mg fresh tissue in the pe-duncle and stem and were not significantly different inthe sweet and bitter variety. In contrast to the samplestaken in April, it was not possible to detect amygdalinin any of the samples taken in May. Upon girdling ofthe stem beneath the first-year shoot and girdling ofthe peduncle, neither prunasin nor amygdalin wasobserved to accumulate in the tissue segments abovethe incision and no decrease was observed below theincision. These results show that transport of cyano-genic glucosides from the shoot apex to the rest of thealmond tree is not the decisive parameter determiningwhether a fruit becomes sweet or bitter. The experi-ments with girdled peduncles strongly indicate thatcyanogenic glucoside accumulation in the bitter al-mond fruit reflects de novo synthesis in the fruit andnot transport from other parts of the plant. To sub-stantiate the latter conclusion, a second type of trap-ping experiments was based on collection of exudatesfrom freshly cut peduncle stubs (Fig. 5). Prunasin aswell as amygdalin was detectable in the exudate, butthe level of amygdalin was again approximately 100-fold lower than that of prunasin. The experimentswere carried out using both a sweet and a bittergenotype, but the genotype did not influence theexuded amounts of cyanogenic glucosides collected.It is difficult to assess how efficient this experimentalsetup is with respect to measuring transport of pru-nasin and amygdalin to the developing fruit. However,

because the girdling experiments with the pedunclesalso provided no indication of transport, we interpretthe two series of experiments to demonstrate thatprunasin and amygdalin are synthesized de novo inthe developing almond fruit.

Measurements of Biosynthetic Activity within theDeveloping Almond Fruit

To investigate which of the tissues in the developingfruit that were biosynthetically active, the differenttypes of tissues were dissected and incubated witheither radiolabeled Phe or with radiolabeled UDPGsupplemented with either mandelonitrile or prunasinas acceptor.

Feeding Experiments with L-[14C]Phe

The sole tissue of the almond fruit that showed theability to convert Phe into prunasin was the tegument(mother tissue). This conversion was observed withteguments from all four genotypes tested (‘Ramillete’[SkSk, sweet], ‘Marcona’ [Sksk, sweet], ‘Garrigues’[Sksk, slightly bitter], and ‘S3067’ [sksk, bitter]; Fig.6). No formation of the cyanogenic diglucoside amyg-dalin was observed. These experiments were carriedout using the freshly dissected thin layer of tegumentcells that upon contact with the air quickly develop abrownish tint. Accordingly, it was not possible to ac-curately quantify the biosynthetic capacity of the dif-ferent genotypes, but several separate experimentsindicate that the capacity for prunasin biosynthesismay be slightly higher in the bitter and slightly bittervarieties compared to the sweet varieties. However,these differences are not major.

Microsome Assays with L-[14C]Phe or L-[U-14C]Tyr

In other cyanogenic plant species, the conversion ofa parent amino acid to the cyanogenic glucoside isknown to be catalyzed by two Cyt P450s anchored inthe membrane system of the endoplasmatic reticulumand by a soluble UDPG glucosyltransferase. Accord-ingly, microsomal preparations harboring the two CytP450s were shown to catalyze the conversion of theparent amino acid into the corresponding cyanohydrin(McFarlane et al., 1975; Fig. 1). When microsomes wereprepared from freshly dissected teguments from thetwo genotypes ‘Ramillete’ (SkSk, sweet) and ‘S3067’(sksk, bitter) and incubated with L-[14C]Phe in thepresence of NADPH, no radiolabeled intermediates orproducts were formed as monitored by radio-thin layerchromatography (TLC; Fig. 7). The expected interme-diates and final product were phenylacetaldoxime,phenylacetonitrile, and mandelonitrile. The mandelo-nitrile is labile and would be detected as the dissoci-ation product benzaldehyde on the TLC. The positionof all these components on the TLC was determinedby coapplication of authentic standards. To assesswhether this negative result reflected the presence of

Figure 5. Prunasin and amygdalin exuded in April from peduncle stubsinto agar and collection of exudate in a septum-covered agar-filledEppendorff tube. [See online article for color version of this figure.]



inhibitory substances in the tegument that interactedwith the Cyt P450s, a new set of experiments werecarried out in which almond teguments or almondroots and 2-d-old sorghum seedlings were homoge-nized together. The microsomes isolated from sorghumseedlings and preparations obtained from coisolationof microsomes from tissues of the two plant specieswere tested for biosynthetic activity with L-[U-14C]Tyr assubstrate (Fig. 7). As expected, the sorghum micro-somes showed strong conversion of L-[U-14C]Tyr into[U-14C]p-hydroxybenzaldehyde, the dissociation prod-uct of p-hydroxymandelonitrile. p-Hydroxymandelo-ntrile is the aglycon of dhurrin, the Tyr-derivedcyanogenic glucoside in sorghum. In contrast, whenthe experiment was repeated with sorghum micro-somes prepared in the presence of almond teguments,no metabolic activity was observed. This documentsthe presence of inhibitory substances in the almondtegument that upon grinding of the plant material are

liberated and inhibit the activity of the sorghum CytP450 enzymes. In comparison, almond roots alsoshowed some inhibitory activity on sorghum micro-somes, but to a much less extent.

[U-14C]UDP-Glucosyltransferase Assays

The distribution of UDPG glucosyltransferase activ-ities able to glucosylate either mandelonitrile (GT1)into prunasin or prunasin into amygdalin (GT2) wasfollowed throughout the growth season from April toJuly with focus on the activities in leaf laminae and inthe different fruit tissues (Fig. 8). These experimentswere carried out by administration of radiolabeled[U-14C]UDPG in combination with unlabeled agluconacceptors to young leaf laminae or tissues dissectedfrom developing fruits. Accordingly, the results ob-tained are not quantitative but indicative of the maindistribution of the two glucosyltransferases. Leaf lam-ina was found to show low UDPG mandelonitrileglucosyltransferase activity over the entire growthphase and independent of the genotype tested. In fruittissues, the activity of this glucosyltransferase wasmore dominant in the bitter compared to the sweetvariety, but activity was indeed observed in mostsamples of the sweet genotype. In contrast to theseresults, UDPG prunasin glucosyltransferase activitywas essentially restricted to the cotyledon with similaractivities in the sweet and bitter variety (Fig. 8). In theexperiments with [U-14C]UDPG and prunasin as theunlabeled acceptor, radiolabeling of prunasin wasalso observed in the cotyledon. This demonstratesthat some of the administered prunasin was de-graded into mandelonitrile by endogenous prunasinhydrolase and the mandelonitrile then reconvertedinto radiolabeled prunasin by the action of UDPGmandelonitrile glucosyltransferase (Fig. 1). Alterna-tively, the source of mandelonitrile could representturnover of the newly formed radiolabeled amygda-lin or of the endogenous pool of amygdalin by thecombined action of amygdalin hydrolase and pruna-sin hydrolase (Fig. 1).

Figure 6. Administration of radiolabeled Phe to excised intact tegu-ment tissue obtained from the four genotypes demonstrating the abilityof all four genotypes to form prunasin.

Figure 7. Analysis of the biosyntheticactivity of microsomes isolated fromteguments and roots of the two geno-types ‘Ramillete’ (SkSk, sweet) and‘S3067’ (sksk, bitter), and demonstra-tion of the presence of Cyt P450 inhib-itors in the almond tegument bycoisolation of microsomes from al-monds and sorghum seedlings.



Localization of b-Glucosidases in the Tegument

Although the tegument of the genotype ‘S3067’ (sksk,bitter) showed a high content of prunasin whereasprunasin was barely detectable in ‘Ramillete’ (SkSk,sweet), the radiolabeling experiments did not indicatemajor differences in biosynthetic capacity between thebitter and sweet genotypes. We therefore focused ourstudy on possible differences in the b-glucosidaseactivity and location in the tegument of the bitter andsweet varieties. b-Glucosidase activity was assessed intransverse sections of the tegument situated adjacentto the developing cotyledon by staining with Fast BlueBB salt in the presence of the b-glucosidase substrate6-bromo-2-napthyl-b-D-glucopyranoside (Fig. 9). Upon1-min staining of sections from ‘Ramillete’ (SkSk,sweet), strong b-glucosidase activity was observed inthe inner epidermis of the tegument facing the nucellus.Higher magnification of the inner epidermis cellsshowed that b-glucosidase staining was restrictedto the cytosol and the main vacuole present in thesecells. When the staining procedure was carried out inthe absence of the b-glucosidase substrate 6-bromo-2-napthyl-b-D-glucopyranoside, no or only very weakstaining was observed. When comparable sections ofthe tegument from ‘S3067’ (sksk, bitter) were analyzedin the light microscope, the presence of the innerepidermis cell layer offering strong staining in thesweet variety was clearly visible. However, this celllayer did not stain with the Fast Blue BB salt together

with the b-glucosidase substrate 6-bromo-2-napthyl-b-D-glucopyranoside, except for the weak backgroundstaining also observed with the sweet variety in theabsence of the b-glucosidase substrate. When the stain-ing period of the sections of ‘S3067’ (sksk, bitter) wasprolonged from 1 to 10 min, a weak b-glucosidaseactivity was detectable. However, this activity wasderived from the apoplast surrounding the inner epi-dermis cell layer, and not from the cytosol and centralvacuole as observed in the sweet genotype.

DISCUSSION

Bitterness in almond is determined by the content ofthe cyanogenic diglucoside amygdalin. It has previ-ously not been clear whether amygdalin accumulationin the bitter kernel reflected transport from other partsof the almond tree or de novo synthesis in the kernel.To address this issue, the occurrence of amygdalinand its precursor prunasin was monitored during theentire growth season from tree flowering to fruitripening with focus on the developing stems, leaflaminae, petioles, and different tissues of the develop-ing fruit. During the entire season, prunasin wasdetected in the vegetative part of all four genotypes‘Ramillete’ (SkSk, sweet), ‘Marcona’ (Sksk, sweet),‘Garrigues’ (Sksk, slightly bitter), and ‘S3067’ (sksk,bitter), but the content was always severalfold higher

Figure 8. A and B, UDPG-mandelo-nitrile glucosyltransferase (GT1; A)and UDPG-prunasin glucosyl trans-ferase (GT2; B) activity monitored bythe formation of radiolabeled pru-nasin and amygdalin upon admin-istration of [U-14C]UDP-Glc to leaflamina, nucellus, and cotyledons of‘Ramillete’ (SkSk, sweet) and ‘S3067’(sksk, bitter) in the presence of man-delonitrile and prunasin, respectively.



in the bitter genotype. In all four genotypes, prunasincontent in leaf laminae, petioles, and stems varied in asimilar manner, probably reflecting the physiologicaldevelopment of the tree. At the beginning of thegrowth season, the content of prunasin increases inleaf laminae and stems. This may be related to mobi-lization of reserves stored in lignified tissues through-out the winter. The prunasin content in stems, petioles,and leaf laminae decreases in the subsequent period(April). This coincides with the initiation of secondarygrowth, formation of terminal shoot sprouts resultingin a significant expansion of the tree crown and withthe formation of the different fruit tissues (exocarp,mesocarp, endocarp, and kernel; Girona and Marsal,1995). Between March and April the fruit reaches max-imum size (Fig. 2) and prunasin levels increase in allfour genotypes although not simultaneously. Previousstudies in several Rosaceae (Seigler, 1981), in otherPrunus species (Selmar et al., 1988) and in almond(Frehner et al., 1990; Dicenta et al., 2002) indicatedsimilar patterns of prunasin and amygdalin accumu-lation although other studies using leaf lamina ofdifferent taxa or hybrids between Prunus species,showed that the ratios of prunasin to amygdalinwere independent of the time of harvest and to varysomewhat erratically (Santamour, 1998). The high but

quite variable differences in prunasin content betweensweet and bitter genotypes indicate that the control ofprunasin level is a polygenic trait (Dicenta et al., 2002).

In contrast to the results with vegetative tissues,prunasin was only detected in the fruits of the bittergenotype (Fig. 3). Most remarkable is the prunasinlevel in the tegument that rises constantly to a level of1.6 mmol/100 g fresh weight at the end of June, wherea rapid decline followed by complete disappearance isobserved. Amygdalin is first detected in the nucellusand endosperm where transient accumulation is ob-served at the end of March. From April, the amygdalinlevel in the cotyledons rises constantly to a level of9 mmol/100 mg fresh weight in the month of Augustwhen the fruit is mature. The sequential appearanceand decline of prunasin level in the tegument andparallel accumulation of amygdalin in the cotyledonsuggested that prunasin produced or imported intothe tegument might serve as a direct precursor foramygdalin formation in the cotyledons.

Girdling experiments using the two genotypes‘Ramillete’ (SkSk, sweet) and ‘S3067’ (sksk, bitter)demonstrated that the prunasin and amygdalin ob-served to accumulate in the tegument and cotyledons,respectively (Fig. 4), were de novo synthesized in thesetissues. In undamaged stems and peduncles, both

Figure 9. Localization of b-glucosidase activ-ity in the inner epidermis of the tegument of‘Ramillete’ (SkSk, sweet) and ‘S3067’ (sksk,bitter) as monitored by staining of thin sections(6 mm) with Fast Blue BB salt in the presenceof a b-glucosidase-specific substrate. A and B,Cross view of the fruit. The areas sectioned forfurther analysis are indicated. A to E, ‘Ram-illete’ (A); ‘S3067’ (B); Fast Blue BB salt stain-ing in the presence of a b-glucosidase-specificsubstrate (C–E). Staining period, 1 min. C to E,‘Ramillete’ (C and D); ‘S3067’ (E). F, ControlFast Blue BB salt staining of ‘Ramillete’ in theabsence of a b-glucosidase-specific substrate.Staining period, 1 min. G to I, Fast Blue BB saltstaining in the presence of a b-glucosidase-specific substrate. G and H, ‘S3067’; stainingperiod, 2 and 10 min, respectively. I, ‘Ram-illete’; staining period, 1 min. c, Cotyledon; ie,inner epidermis of the tegument; n, nucellus;t, tegument. Bars and the width of G to Icorrespond to 100 mm.



prunasin and amygdalin were detected in low butvarying amounts. The prunasin content was alwayshigher in the ‘S3067’ (sksk, bitter) compared to‘Ramillete’ (SkSk, sweet), whereas the amygdalin con-tent was about the same in the bitter and sweet geno-type. In no case did analysis of stem internodes beneaththe first-year shoots and of peduncle segments aboveand below the incisions show significant changes in thecontent of prunasin and amygdalin. Likewise, in thesap exuded from freshly cut peduncles, no differencesin the amounts of prunasin and amygdalin betweenthe sweet and bitter genotype was observed (Fig. 5).This strongly argues that the prunasin and amygdalinpresent in the fruit is formed by de novo synthesis inthe fruit in course of fruit development. Similar gir-dling experiments have previously been carried out incassava and demonstrated the significance of trans-port of cyanogenic glucosides from the site of produc-tion in the actively growing shoot apexes and leaflaminae to the tuber. Thus in cassava, the cyanogenicglucoside content in the internode above the incisionzone in the shoot apex increased by a factor of 75(Jørgensen et al., 2005). This clearly demonstrates thefeasibility of using the girdling method to assesstransport of cyanogenic glucosides.

A number of experiments were carried out to assessin which fruit tissues the synthesis of prunasin andamygdalin takes place. Upon administration of radio-labeled Phe to different excised tissues of the fruit, theonly tissue that showed capacity to produce radiola-beled prunasin was the tegument. No major differ-ences in the ability of the four tested sweet, slightlybitter, and bitter genotypes to produce radiolabeledprunasin was observed (Fig. 6). To obtain a quantita-tive measure of the biosynthetic capacity, microsomeswere prepared from each of the four genotypes. Un-fortunately, this approach was negative, because of thepresence of a Cyt P450 inhibitor in the tegument tissuethat completely inactivated the microsomal prepara-tions as shown by cohomogenization of the almondteguments with sorghum seedlings that actively syn-thesize dhurrin. This resulted in inactivation of thesorghum Cyt P450 enzyme system (Fig. 7). We have pre-viously reported that the tegument in cassava seedscontains a potent Cyt P450 inhibitor (Koch et al., 1992)and that this also applies to the seed coat of sorghum(Halkier and Møller, 1989). The physiological role ofthis inhibitory activity is currently not understood butmay serve to block detrimental production of cyano-genic compounds that would end up being degradedwith concomitant release of HCN when the tegumentdries out during the final stages of fruit ripening. Inaddition to the relevant Cyt P450 enzymes, prunasinformation in the tegument would also require the pres-ence of UDPG mandelonitrile glucosyltransferase activ-ity (UGT1; Fig. 1). Radiolabeling experiments detectedthis activity in leaf laminae as well as in all differentfruit tissues and the activity was higher in fruit tissuesof the bitter genotype ‘S3067’ compared to those of thesweet genotype ‘Ramillete’ (Fig. 8). These data identify

the tegument as the site of prunasin synthesis in thedeveloping almond.

In cassava, the two cyanogenic glucosides linamarinand lotaustralin contribute to the bitterness of the tu-bers. However, other bitter constituents like isopropyl-b-D-apiofuranosyl-(1/6)-b-D-glucopyranoside are alsomain contributors to the bitterness (King and Bradbury,1995). In almonds, bitterness is essentially determinedby their content of amygdalin (Dicenta et al., 2002).When the amygdalin content reaches very high levelsas in the bitter genotypes, the sense of taste cannot beused to assess the level of bitterness. According toRemaud et al. (1997), the benzaldehyde produced uponhydrolysis of amygdalin is responsible for the bitter-ness in almonds. The flavor of benzaldehyde can beappreciated even upon chopping almonds with a lowamygdalin content (the slightly bitter genotypes). Someslightly bitter genotypes do vary their flavor depend-ing on the environmental conditions (Dicenta et al.,2007) but glycosides unrelated to cyanogenic glyco-sides have not yet been shown to contribute to bit-terness in almonds.

Amygdalin is formed from prunasin by the action ofa UDPG prunasin glucosyltransferase (Fig. 8). Radio-labelling experiments demonstrated that this enzymeactivity was restricted to the nucellus, endosperm, andcotyledon with by far the strongest activity observedin the cotyledons. This would imply that prunasinsynthesized in the tegument is transported to thenucellus, endosperm or cotyledon where it becomesglucosylated and finally is stored in the cotyledon.Amygdalin only accumulates in the bitter genotype‘S3067’ and this matches very well the observed spe-cific accumulation of the precursor prunasin in thetegument of ‘S3067’. But from the radiolabeling exper-iments, the sweet genotypes would also be predictedto be able to produce amygdalin because all biosyn-thetic enzyme activities were present, except for theUDPG mandelonitrile glucosyltransferase in amountssimilar to those found in the bitter genotype. Thisprompted us to investigate whether the differencebetween the ability of the bitter and sweet genotypesto accumulate amygdalin resided in their ability totransfer the prunasin precursor molecule from thetegument to the nucellus, endosperm, or cotyledon.This issue was addressed by studying thin sections ofwhole seeds (tegument, nucellus, endosperm, andcotyledon) at the developmental stage where prunasincontent declined in the tegument of the bitter geno-type. Thin sections (6 mm) were stained with Fast BlueBB salt together with the b-glucosidase substrate6-bromo-2-napthyl-b-D-glucopyranoside to monitorb-glucosidase activity. In the sweet variety ‘Ramillete’,the staining showed the existence of a continuousb-glucosidase-rich cell layer in the inner epidermis ofthe tegument, facing the nucellus. The b-glucosidaseactivity in these cells was restricted to the cytoplasmand the main central vacuole. The similar cell layer wasalso present in the bitter genotype but in this genotypestaining with the Fast Blue BB salt together with the



b-glucosidase substrate was weak and was restrictedto the apoplastic space. If transport of prunasin fromtegument to cotyledon proceeds via the symplast,this would indicate that the prunasin synthesized inthe tegument of the sweet variety (SkSk) is degradedwhen it has to pass this cell layer. The tegument is thethin layer of mother-derived tissue that encapsulatesthe nucellus and the developing endosperm and em-bryo and this mechanism for control of bitternesswould comply with the notion that the genetic controlof this trait is mother-tissue dependent (Werner andCreller, 1997; Dicenta et al., 2007) and the gene isrecessive (Heppner, 1923, 1926; Dicenta and Garcıa,1993; Dicenta et al., 2007).

But why would prunasin first be synthesized in thetegument tissue of the developing almond fruit in thesweet variety and subsequently be subjected to deg-radation? In sorghum seedlings, the Cyt P450 systemcatalyzing dhurrin synthesis constitutes about 0.5%of the total membrane proteins (Sibbesen et al., 1994),yet the cyanogenic glucoside dhurrin accounts for 30%of the dry weight in the apex of the sorghum seedling(Halkier and Møller, 1989). It is possible that in almonda similarly active Cyt P450 system could convertexcess free Phe transported to or synthesized withinthe tegument into prunasin. Degradation of the pru-nasin by the action of the cyanogenic b-glucosidasewould then result in the release of HCN, which, by theaction of b-cyano-Ala synthase, would first be con-verted into b-cyano-Ala and subsequently convertedinto Asn and Asp. Recently a different pathway forcyanogenic glucoside catabolism involving nitrilaseheterodimers has been suggested to operate in sor-ghum (Jenrich et al., 2007; Kriechbaumer et al., 2007).In this pathway, the nitrogen atom of the nitrile groupis recovered into ammonia without intermittent re-lease of toxic HCN. The two pathways offer theopportunity to convert Phe into ammonia, Asp, andAsn, which could be used as general precursors foramino acid and protein synthesis in the developingcotyledons. The sweet almond varieties may thus profitby having a more balanced and alternative direct sup-ply of free amino acids for protein synthesis in thedeveloping cotyledons whereas the bitter variety ac-cumulating amygdalin in the cotyledons may profitfrom the protection offered by this secondary metab-olite toward herbivores and pests. In the bitter geno-type that accumulates amygdalin, this opportunity ofusing the secondary metabolite as a buffer for primarymetabolism has been retained. But in contrast to thesituation in the sweet variety, this opportunity is ex-ploited when the amygdalin-containing seed is readyto germinate and the amygdalin stored in the cotyle-dons is turned over as happens within a 3-week periodfor 80% of the amygdalin stored in black cherry seeds(Swain and Poulton, 1994b). Thus cyanogenic gluco-side production and accumulation in almonds appearsto constitute yet another example where formation of asecondary plant product serves to balance processes inprimary metabolism by providing a buffer capacity.

Cassava contains the two cyanogenic glucosideslinamarin and lotaustralin (Andersen et al., 2000). Inthis plant species, differences in b-glucosidase locationhave also been shown between cultivars with low andhigh in cyanogenic glucoside content (Santana et al.,2002). In the cultivar accumulating high amounts ofcyanogenic glucosides, the cyanogenic b-glucosidase(linamarase) was immunolocalized to the cell wall andcytosol. In the cassava cultivar low in cyanogenicglucosides, linamarase was mainly located in modifiedvacuoles of laticifer cells and in the cytosol of paren-chyma cells. This parallels our observations in bitter(sksk) and sweet (SkSk) almonds. In this study wehave not directly shown that the cell layer in the teg-ument high in b-glucosidase activity actually containsthe cyanogenic b-glucosidase. In classic cytology, theFast Blue BB salt is typically used as an unspecific stainfor b-glucosidases and esterases. However, as used inthis study, we consider the staining method fairlyspecific for cyanogenic b-glucosidases. Thus stainingof cross sections of leaves of Arabidopsis, which donot contain a cyanogenic b-glucosidase produced nostaining when carried out with the short stainingperiod of 1 min used in this study. In contrast, strongstaining was observed with transgenic Arabidopsisplants expressing two cyanogenic b-glucosidases fromLotus japonicus (A.V. Morant, unpublished data).

Bitterness in almond is inherited as a single recessivegene (sksk, bitter) and controlled by the genotype ofthe seed mother (Heppner, 1923; Kester and Gradziel,1996; Dicenta and Garcıa, 1993; Socias i Company,1998). The genetic basis has recently been confirmedin crosses of two homozygous bitter genotypes, whichresulted in descendants that were all bitter (Dicentaet al., 2007). Since 1830, the bitter principle in almondshas been known to be amygdalin (Lechtenberg andNahrstedt, 1999). But what then is the genetic basis forthe existence of slightly bitter genotypes? The slightlybitter genotypes have been proposed to be heterozy-gous although not all the heterozygous individualshad a slightly bitter flavor (Dicenta and Garcıa, 1993).It was also observed that when heterozygous slightlybitter genotypes, e.g. ‘Garrigues’, were crossed, ahigher proportion of slightly bitter seedlings was ob-tained in comparison to crosses carried out with hetero-zygous sweet cultivars, e.g. ‘Marcona’. We hypothesizethat the Sk gene encodes prunasin hydrolase and thatthe fluctuating degree of bitterness in the heterozy-gotes may be related to the detection of b-glucosidaseactivity in two subcellular compartments in the ho-mozygous sweet variety (SkSk), namely the cytosoland the central vacuole. Because bitterness is inheritedas a single recessive gene (sksk, bitter), this impliesthat the single gene harbors multiple transcription ini-tiation sites. These may give rise to two types of mRNApossessing two in-frame ATG codons for translationinitiation and encoding two isoforms with differenttargeting information. One isoform would remain in thecytosol whereas the other isoform would be destinedfor the vacuole. Alternative transcription initiation is



known for other plant genes (Soll, 1998; Obara et al.,2002). According to this hypothesis, amygdalin accu-mulation would then be quite variable in the heterozy-gous genotypes (Sksk) as a result of varying proportionsof b-glucosidase localized inside or outside the vacu-ole. This is in full agreement with the experimentalobservations. Amygdalin content is constantly high inthe homozygous (sksk) bitter genotypes but fluctuatesfrom zero to medium levels in the heterozygous geno-types (Arrazola, 2002; Dicenta et al., 2002). Work is inprogress to achieve biochemical and molecular char-acterization of the b-glucosidase present in the tegu-ment of the sweet almond genotypes and hopefully toutilize the gene encoding this b-glucosidase as a tool inmarker-assisted selection against bitter almonds.

MATERIALS AND METHODS

Plant Material

Almond (Prunus dulcis) branches of the following four genotypes ‘Ramillete’

(SkSk, sweet), ‘Marcona’ (Sksk, sweet), ‘Garrigues’ (Sksk, slightly bitter), and

‘S3067’ (sksk, bitter) were provided by the Almond Breeding Program of

Centro de Edafologıa y Biologıa Aplicada del Segura-Consejo Superior de

Investigaciones Cientıficas (CEBAS-CSIC). Every second week during the

growth season from tree flowering to kernel maturity (March to August), plant

material was sent to the Plant Biochemistry Laboratory at the University of

Copenhagen by courier shipment. The branches were placed in water and

used as a source of leaf laminae, petioles, stems, peduncles, roots, and fruits.

Fruits were separated into mesocarp, endocarp, tegument, nucellus, endo-

sperm, and cotyledon as required for the different sets of experiments. All

analyses were carried out on the day of arrival of the plant material, i.e. the

day after the branches were cut off the tree. Roots analyzed were from 9-year-

old plants. Girdling experiments were carried out using almond trees growing

in the orchard at CEBAS-CSIC. Sorghum seeds (Sorghum bicolor ‘SS1000’) were

purchased from Agripro.

LC-MS Analysis of Cyanogenic Glucoside Content

Plant material (leaf lamina, petiole, stem, tegument, nucellus plus endo-

sperm, and cotyledon; three sample specimens of each) was weighed and

immersed in boiling MeOH (80%, 500 mL, 5 min). The material was ground

with a small pestle and filtered (0.22 mm low-binding Durapore membrane)

after addition of lotaustralin (10 mg) as an internal standard. Analytical LC-MS

was carried out on an Agilent 1100 Series LC (Agilent Technologies) coupled

to a HCTplus ion trap mass spectrometer (Bruker Daltonics). The column was

a Synergy Fusion-RP column (Phenomenex; 2.5 mm, 100 A, 2 3 50 mm), and

the flow rate was 0.3 mL min21. The mobile phases were as follows: (1) 0.1%

(v/v) formic acid and 50 mM NaCl in water; and (2) 0.1% (v/v) formic acid in

acetonitrile. The gradient program was as follows: (1) 0 to 7.5 min, linear

gradient 6% to 19% (v/v); and (2) 7.5 to 10 min, linear gradient 19% to 100%.

The mass spectrometer was run in positive ion mode. Traces of total ion

current and of extracted ion currents for specific [M 1 Na]1 adduct ions were

used to identify peaks. The retention time for lotaustralin, amygdalin, and

prunasin was 1.9, 4.1, and 4.8 min, respectively.

Girdling Experiments

Phloem transport of cyanogenic glucosides in peduncles of developing

fruits and in the stems beneath the first-year shoots was monitored in girdling

experiments. The epidermis and cambium cell layers including the phloem

were removed by scalpel incisions (2-mm wide). The experiments (five

replicates) were carried out in April and May using almond trees of the

genotype ‘Ramillete’ (SkSk, sweet) and ‘S3067’ (sksk, bitter) growing at the

experimental field at CEBAS-CSIC. Three days after the girdling took place,

the sections positioned above and below the incision site (0.5-cm peduncle

segments; 1.0-cm stem segments) were excised and boiled separately in

MeOH (80%, 5 min). After filtering (0.22 mm low-binding Durapore mem-

brane), the prunasin and amygdalin content of the MeOH extract was deter-

mined by LC-MS. As a reference (two replicates), corresponding segments

were excised from peduncles and stems that had not been girdled.

In parallel to the girdling experiments, the peduncles of developing fruits

were cut and each of the peduncle stubs remaining on the tree were im-

mediately immersed into a septum-covered Eppendorf tube filled with an

agar (0.9%, w/v)/EDTA (20 mM, pH 6.0). After 3 d (five replicates), the

cyanogenic glucoside content in the agar was extracted in MeOH (80%) and

measured using LC-MS as previously described.

Tissue Biosynthetic Activity

Leaf laminae, petioles, stem, tegument, nucellus plus endosperm, and

cotyledons from the almond genotypes ‘Ramillete’ (SkSk, sweet) and ‘S3067’

(sksk, bitter) were obtained at different developmental stages throughout

March to May and incubated in L-[14C]Phe (0.125–1.25 mCi, 321 mCi/mmol;

Amersham Biosciences), NADPH (0.1 mM), and dithiothreitol (DTT; 1 mM). At

the end of the incubation period (12 h, 20�C), the material was immersed in

boiling MeOH (80%, 500 mL, 5 min), filtered (0.22-mm low-binding Durapore

membrane), and aliquots (10 mL) were applied to silica gel 60 F254 TLC plates

(Merck). Radiolabeled cyanogenic glucosides formed were separated by

development in EtOAc/HOAc/MeOH/H2O (8:2.5:2.5:1, v/v) and monitored

using a Storm 860 PhosphorImager (Molecular Dynamics). The position of

prunasin and amygdalin was defined by the UV absorption of coapplied

unlabeled authentic standards.

Microsomal Preparations

Plant material (3–100 g fresh weight of leaf laminae, roots, and fruit

exocarp, endocarp, and mesocarp, tegument, nucellus plus endosperm, and

cotyledon) from the genotypes ‘Ramillete’ (SkSk, sweet) and ‘S3067’ (sksk,

bitter) was harvested in April and May and homogenized with 0.1 mass of

polyvinylpolypyrrolidine in a buffer composed of 250 mM Suc, 100 mM Tricine

(pH 7.9), 50 mM NaCl, 2 mM EDTA, and 2 mM DTT using mortar and pestle or

mechanical food chopper as required. The homogenate was filtered through a

nylon cloth (50-mm mesh) and centrifuged (10 min, 12,000 rpm, 4�C). Micro-

somes were recovered from the supernatant by centrifugation (60 min, 46,000

rpm) and resuspended in 50 mM Tricine (pH 7.9)/2 mM DTT. Microsomes were

incubated (30 min, 30�C, total volume 20 mL) with 0.05 mCi L-[14C]Phe (321

mCi/mmol; Amersham Biosciences) in the presence or absence of 1 mM

NADPH. An aliquot (10 mL) was applied to the silica gel 60 F254 TLC plates

and radiolabeled metabolites formed were separated by development in

toluene/EtOAc (5:1, v/v) and monitored using the Storm 860 Phosphor-

Imager. Phenylacetaldoxime, phenylacetonitrile, and benzaldehyde were ap-

plied to the TLCs as reference compounds and their position located by their

UV absorbance.

To analyze whether the almond plant material was containing inhibitors of

the microsomal Cyt P450 system, microsomes were prepared from cohomog-

enized 3-d-old etiolated sorghum seedlings (12 g) and almond tissue (roots

and teguments; 3 g) of the genotypes ‘Ramillete’ and ‘S3067’. In these

experiments, microsomes were incubated using 0.05 mCi L-[U-14C]Tyr (443

mCi/ mmol; Amersham Biosciences) as substrate. A separate microsomal

preparation from sorghum was used as a control. p-Hydroxyphenylacetal-

doxime, p-hydroxyphenylacetonitrile, and p-hydroxybenzaldehyde were used

as reference compounds (Møller et al., 1977; Møller and Conn, 1979).

Glucosyltransferase Assays

Enzyme extracts from leaf laminae, petioles, peduncles, nucellus plus

endosperm, and cotyledons (100- to 500-mg tissue) of the following four

genotypes ‘Ramillete’ (SkSk, sweet), ‘Marcona’ (Sksk, sweet), ‘Garrigues’

(Sksk, slightly bitter), and ‘S3067’ (sksk, bitter) were prepared every second

week throughout the entire growth season (March to August) by homogeni-

zation (Eppendorff tube, pestle, 4�C) in 250 mM Suc, 100 mM Tris-HCl (pH 7.5),

50 mM NaCl, 2 mM EDTA, 5% (w/v) polyvinylpolypyrrolidone, 200 mM

phenylmethylsulfonyl fluoride, and 6 mM DTT (total volume, 0.5 mL). The

supernatants were collected after centrifugation (20,000g, 20 min) and aliquots

(5 mL) were incubated (total volume, 20 mL) with 20 mM acceptor (prunasin or

mandelonitrile), 0.025 mCi [U-14C] UDPG (200 mCi/mmol; Amersham Bio-

sciences), and 25 mM d-gluconolactone (b-glucosidase inhibitor) in 100 mM

Tris-HCl (pH 7.5). After incubation (10 min, 30�C), aliquots (10 mL) were



applied to the silica gel 60 F254 TLC plates and radiolabeled products

separated by development EtOAc/HOAc/MeOH/H2O (8:2.5:2.5:1, v/v) and

monitored using the Storm 860 PhosphorImager. The position of prunasin and

amygdalin on the TLCs was defined by coapplication of the unlabeled

authentic standards.

Tissue Sections and b-Glucosidase Staining Using FastBlue BB Salt

In April, fruit samples (tegument, nucellus, and cotyledon) from the

genotypes ‘Ramillete’ and ‘S3067’ were imbedded in plastic according to the

manufacturers manual for Technovit 8100 (Heraeus) with minor alterations.

The tissues were dehydrated in a graded series of acetone solutions (25%, 50%,

and 100%, v/v, 1 h each) and left overnight in the filtration solution. Sections

(6 mm) were cut on a Reichert-Jung 2030 rotary microtome (Reichert-Jung).

The sections were stained for different time periods (1–10 min) with Fast

Blue BB salt with and without the substrate 6-bromo-2-napthyl-b-D-gluco-

pyranoside (Spielman and Mowshowitz, 1982) to detect b-glucosidase activity in

the cells.

ACKNOWLEDGMENTS

We thank Teresa Cremades Rosado and Mariano Gambın for technical

help.

Received November 13, 2007; accepted December 29, 2007; published January

11, 2008.

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Bitterness in Almonds1[C][OA] · mation in plants has its origin in antiquity. In ancient Egypt, traitorous priests in Memphis and Thebes were poisoned to death with pits of peaches