Prunasin hydrolases localization during fruit development in sweet and bitter almonds

18 Pages·2017·4.26 MB·English

university of copenhagen
Prunasin hydrolases localization during fruit development in sweet and bitter almonds
Sánchez Pérez, Raquel; Belmonte, Fara Sáez; Borch-Jensen, Jonas; Dicenta, Federico;
Møller, Birger Lindberg; Jørgensen, Kirsten
Published in:
Plant Physiology
DOI:
10.1104/pp.111.192021
Publication date:
2012
Document version
Publisher's PDF, also known as Version of record
Citation for published version (APA):
Sánchez Pérez, R., Belmonte, F. S., Borch-Jensen, J., Dicenta, F., Møller, B. L., & Jørgensen, K. (2012).
Prunasin hydrolases localization during fruit development in sweet and bitter almonds. Plant Physiology, 158(4),
1916-1932. https://doi.org/10.1104/pp.111.192021
Download date: 22. Feb. 2023
Prunasin Hydrolases during Fruit Development
in Sweet and Bitter Almonds1[C][W][OA]
Raquel Sa´nchez-Pe´rez, Fara Sa´ez Belmonte, Jonas Borch, Federico Dicenta,
Birger Lindberg Møller*, and Kirsten Jørgensen
Department of Plant Breeding, Centro de Edafolog´ıa y Biolog´ıa Aplicada del Segura-Consejo Superior de
Investigaciones Cient´ıficas, E–30100 Espinardo, Murcia, Spain (R.S.-P., F.D.); Plant Biochemistry Laboratory,
Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Copenhagen, Denmark (R.S.-P., B.L.M.,
K.J.); Department of Bioimaging, Campus Universitario de Espinardo, 30100 Murcia, Spain (F.S.B.); and
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK–5230 Odense M,
Denmark (J.B.)
Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin
concentrationincreasesinthecourseoffruitformation.Themonoglucosideprunasinistheprecursorofamygdalin.Prunasin
maybedegradedtohydrogencyanide,glucose,andbenzaldehydebytheactionoftheb-glucosidaseprunasinhydrolase(PH)
andmandelonitirilelyaseorbe glucosylatedtoformamygdalin.The tissueandcellularlocalizationofPHswasdetermined
duringfruitdevelopmentintwosweetandtwobitteralmondcultivarsusingaspecificantibodytowardPHs.Confocalstudies
onsectionsoftegument,nucellus,endosperm,andembryoshowedthatthelocalizationofthePHproteinsisdependentonthe
stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two
different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86%
identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692
display92%and86%nucleotideidentitytoPh1fromblackcherry(Prunusserotina).Bothproteinswerepredictedtocontainan
amino-terminalsignalpeptide,withthesizeof26aminoacidresiduesforPH691and22residuesforPH692.ThePHactivity
and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different
concentrationsofprunasinavailableintheseedforamygdalinsynthesisandthatthesedifferencesmaydeterminewhetherthe
maturealmond developsinto bitteror sweet.
Prunasin isa cyanogenic monoglucoside present in is derived from the amino acid Phe (Mentzer and
almond (Prunus dulcis; syn. Prunus amygdalus) and is Favre-Bonvin,1961;Ben-YehoshuaandConn,1964).The
foundinmosttissueparts,includingroots,shoots,and pathwayforprunasinbiosynthesisincludesphenylace-
unripe fruits (Frehner et al., 1990; Arra´zola, 2002; taldoxime, phenylacetonitrile, and mandelonitrile as
Dicentaetal.,2002;Wirthensohnetal.,2008).Prunasin intermediates. In analogy with studies in other cyano-
genicplantspecies,thispartofthepathwayisthoughtto
be catalyzed by two multifunctional membrane-bound
1ThisworkwassupportedbytheDanishCouncilforIndependent cytochrome P450 enzymes (Sa´nchez-Pe´rez et al., 2008),
Research,Technology,andProductionSciences,theVillumFoundation althoughthesegeneshavenotyetbeencharacterizedin
totheresearchcenter“Pro-ActivePlants,”andtheCenterofSynthetic almond. The last step in the pathway involves the con-
Biology funded by the UNiversitetsforskningens InvesteringsKapital versionofmandelonitrileintoprunasinandiscatalyzed
researchinitiativeoftheDanishMinistryofScience,Technology,and
byasolubleUDP-glucosyltransferaseUGT85A19(Franks
Innovation (to B.L.M. and K.J.), by the Seneca Foundation (project
etal.,2008).
Biolog´ıaMoleculardelaCianoge´nesisenAlmendro)andtheSpanish
Biosynthetic studies in developing fruits based on
Ministry of Science and Innovation (project Mejora Gene´tica del
the administration of radiolabeled precursors to ex-
Almendro;toR.S.-P.andF.D.),andbyapostdoctoralfellowshipfrom
theMinistryofEducationandScienceandaJuntaparalaAmpliacio´n cisedtissuesdemonstrated prunasinsynthesistopro-
de Estudios de Doctores contract from the Consejo Superior de ceedinthetegumentcells,withsimilarratesinsweet
InvestigacionesCient´ıficas,Spain(toR.S.-P.). and bitter cultivars. In neither sweet nor bitter culti-
*Correspondingauthor;[email protected]. varsdidthetegumenttissue,alsocalledinteguments,
Theauthorresponsiblefordistributionofmaterialsintegraltothe testa,orseedcoat(HawkerandButtrose,1980;Dourado
findings presented in this article in accordance with the policy et al., 2004), support amygdalin synthesis (Sa´nchez-
describedintheInstructionsforAuthors(www.plantphysiol.org)is: Pe´rezetal.,2008).Atransientaccumulationofpruna-
BirgerLindbergMøller([email protected]).
sin in the tegument of bitter almond cultivars was
[C]Somefiguresinthisarticlearedisplayedincoloronlinebutin
observed, whereas no such accumulation was seen in
blackandwhiteintheprintedition.
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. sweetcultivars(Sa´nchez-Pe´rezetal.,2008).Following
[OA]OpenAccessarticlescanbeviewedonlinewithoutasubscription. thetransientaccumulationofprunasininthetegumentof
www.plantphysiol.org/cgi/doi/10.1104/pp.111.192021 thebittercultivars,amygdalinwasobservedtogradually
1916 Plant Physiology(cid:1), April 2012, Vol.158, pp. 1916–1932, www.plantphysiol.org (cid:3)2012AmericanSociety of Plant Biologists. AllRights Reserved.
Downloaded from www.plantphysiol.org on February 16, 2015 - Published by www.plant.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Prunasin Hydrolases in Sweet and Bitter Almonds
accumulate in the nucellus and endosperm in the mid Piotrowski et al., 2001; Piotrowski and Volmer, 2006;
developing stages. Once almonds are fully ripened, the Jenrich et al., 2007; Kriechbaumeret al., 2007).
only quantitative important site of accumulation is the Recent studies of bitterness in almond showed a
embryo of the bitter cultivars. In comparison, the hightotalb-glucosidaseactivityintheinnerepidermis
amounts of amygdalin detectable in sweet cultivars are ofthetegumentinsweetcomparedwithbitteralmond
minute(Sa´nchez-Pe´rezetal.,2008,Fig.3). cultivars that might interfere with and decrease pru-
The conversion of prunasin to amygdalin requires nasinconversionintoamygdalin(Sa´nchez-Pe´rezetal.,
theactionofayetunidentifiedUDP-glucosyltransfer- 2008). Depending on the cellular localization of the
ase (Frehner et al., 1990; Sa´nchez-Pe´rez et al., 2008; PH activity (apoplastic, cell wall bound, vesicular, or
Wirthensohn et al., 2008). Biosynthetic studies based cytosolic) and whether the route of transport of
on the administration of radiolabeled UDPG in the prunasinfromthebiosyntheticcellsofthetegumentto
presence of prunasin showed that the amygdalin- the nucellus, endosperm, or embryo takes place in the
forming glucosyltransferase activity was essentially symplast or in the apoplast, the b-glucosidase activity
limited to the cotyledons, with residual activities might control the amount of prunasin available for
detected in the nucellus and endosperm. Notably, the amygdalinproduction(Sa´nchez-Pe´rezetal.,2008).
amygdalin-formingglucosyltransferaseactivitywasat Inthisstudy,thelocalizationandactivity ofPHsin
thesamelevelinthesweetandbittercultivarsstudied. different seed tissues were monitored in two sweet
Amygdalin accumulation is also observed in other andtwobitteralmond cultivars duringfruit develop-
Rosaceae seeds, such as in apples (Malus domestica), menttoinvestigateapossiblecorrelationbetweenthe
apricots (Prunus armenaica), black cherries (Prunus content of amygdalin in the almond kernel and the
serotina), peaches (Prunus persica), and plums (Prunus cellular localizationof PHs.
domestica; McCarty et al., 1952; Conn, 1980; Frehner
etal.,1990;MøllerandSeigler,1991;Swainetal.,1992;
Poulton andLi, 1994). RESULTS
Amygdalin constitutes the bitter principle in bitter
PHLocalizationasMonitoredUsingtheSugar-Reducing
almonds. The avoidance of bitterness in almond ker-
Assay and Antibodies in Unripe Almond Seeds
nels is the main trait considered by almond breeders.
Its single inheritance was discovered by Heppner ThelocalizationofPHactivityinthinsectionsofthe
(1923), who stated that the bitter phenotype is reces- sweetalmondcultivarLauranneandthebitteralmond
sive.Thissinglegene,calledSweetkernel(Sk),hasbeen cultivar S3067 at 154 Julian days (JD; the number of
localized in linkage group 5 (out of eight) of two daysafterJanuary1;Fig.1)wasmonitoredcolorimet-
almond linkage maps (Joobeur et al., 1998; Sa´nchez- rically (red color formation) by the release of Glc
Pe´rez et al., 2007). Although molecular markers for following incubation with prunasin (Sa´nchez-Pe´rez
bitterness have been added close to the Sk locus etal.,2009).Atthisstage,thenucellusandendosperm
(Sa´nchez-Pe´rez et al., 2010), the sequence of this gene weredifficulttoseparatefromthetegument;therefore,
remains unknown. these areanalyzed as a single combined sample. This
Because biosynthetic studies had indicated that (1) also applies to the PH activity experiments (see be-
the capacity to synthesize prunasin in the tegument low). The presence of PH was confined to small
tissue of sweet and bitter almond cultivars appeared vesicles in both cultivars, as judged by the staining
similar and (2) the activity of the glucosyltransferase pattern observed (Fig. 1, A and B) when compared
converting prunasin to amygdalin in the developing withcontrolsamples(Fig.1,EandF).Inboththebitter
embryos was also similar in sweet and bitter almond and sweet cultivars, vesicles in the tegument tissue
cultivars, different rates of prunasin hydrolysis or layerstainedstrongly,whileonlyafewvesiclesinthe
turnover inthedevelopingfruits maydefine whether nucellawerefoundtoreact.Intheendosperm,aslight
their kernels are bitter or sweet. Prunasin may be reddish coloration was observed, reflecting the back-
hydrolyzed into mandelonitrile and Glc by the action ground reaction seen in this tissue. The strongest
of prunasin hydrolases (PHs), specific b-glucosidases reaction was detected in the inner epidermis of the
belongingtofamily-1b-glycosidases(EC3.2.1.21).Ina tegument and may represent the presence of a high
similarmanner,amygdalinmaybehydrolyzedbythe amount of PH enzyme, the presence of PH with
actionofamygdalinhydrolasesintoprunasinandGlc increased specific activity, or the presence of more
(EC 3.2.1.117). The mandelonitrile formed may disso- than one or adifferentisoform in thistissue.
ciateintobenzaldehydeandhydrogencyanide(HCN) InordertodirectlymonitorthelocalizationofthePH
nonenzymaticallyorcatalyzedbymandelonitrilelyase protein,aparallelseriesofexperimentswasperformed
1(EC4.1.2.10;SwainandPoulton,1994a;Suelvesand usinganantibodyknowntospecificallyrecognizePH.
Puigdome`nech, 1998). In a putative alternative path- Thesestudieswerecarriedoutusingtissuesectionsat
way incorporating the action of heteromeric NIT4 thesamedevelopmentalstagesasusedfortheactivity
nitrilases and additional, hitherto unidentified en- stain-based experiments (Sa´nchez-Pe´rez et al., 2009;
zymes, prunasin may be degraded into benzoic acid, Fig.2).PHwasimmunolocalizedtospecificvesiclesas
ammonia, and Glc and in this manner be redrawn visualizedbygreenfluorescence(Fig.2),alocalization
into primary metabolism (Swain and Poulton, 1994b; beingconsistentwiththevesicle-specificlocalizationof
Plant Physiol. Vol. 158, 2012 1917
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Sa´nchez-Pe´rez et al.
seeds of two sweet (Ramillete and Lauranne) and
two bitter (S3067 and Gorki) cultivars was monitored
during fruit development using a specific antibody
towardPHs(Figs.3and4).Becauseofcultivardiffer-
encesinfloweringtimeandseed-ripeningperiod,the
thickness of the endosperm and the shape of the
endosperm cells were not directly comparable when
cultivars were analyzed at the same JD, but compar-
ison of seeds of the same development stage was
possible because of thesequential harvesting.
PH in the Tegument
PHs werepresentin vesicles in thevast majority of
cells in the tegument and were most abundant in the
inner epidermis of the tegument at 154 JD (Fig. 3). At
thelaterstagesofseeddevelopment(from182JD),the
tegument collapses and dries out concomitant with a
reduced content of PH. The inner epidermis of the
tegument showed strong staining especially at the
young stages, in a similar manner to that observed
with the distribution of PH activity and the antibody
stainingpatternsshowninFigures1and2.Thisshows
the presence of high concentrations of PH enzyme in
this cell layer. The differences in localization between
the cultivars are most clearly visualized at higher
magnifications(Fig.3, columns b, d,ande).
PH in the Endosperm
PH was not detected in all individual cells of the
Figure1. Seedsfromtwoalmondcultivars,sweet(Lauranne;AandC) endosperm. In the two bitter cultivars, a distinct PH-
andbitter(S3067;BandD),at154JDwerecross-sectionedtomonitor staining zone was found in the endosperm cells adja-
the distribution of PH activity using the sugar-reducing assay after
cent to the inner epidermis of the tegument. In the
incubationwithprunasin.AandB,PHwasdetectedinthetegument(t),
two sweet cultivars, a more uniform presence of PH-
innerepidermisofthetegument(iet),nucellus(n),andendosperm(en),
containing cells was observed across the endosperm.
as observed by reddish vesicles indicated with arrows. C and D,
Inallfourcultivarsexamined,PHwasobservedinthe
Histologicaldiagrams,definingthedifferenttypeoftissuesanalyzedin
the cross-sections. E and F, Background staining in the absence of symplast of the cells, as shown by three-dimensional
added substrate in Lauranne (E) and S3067 (F). Bars = 50 mm. [See images(Fig.3, last column, 182JD3D).
onlinearticleforcolorversionofthisfigure.]
PHs in theCotyledon of theEmbryo
the PH activity (Fig. 1). PH-containing vesicles were
observed in cells of the endosperm, nucellus, and the The localization of PH proteins in the developing
tegument tissue layer in both cultivars (Fig. 2, A and cotyledonswasfollowedfrom105to220JD(Fig.4).The
D). A higher magnification of the inner epidermis of localization was found to vary over the course of seed
the tegument revealed that PH was exclusively local- development in a manner characteristic for each culti-
izedinthesymplastoftheinnerepidermistissuelayer var.Differencesspecifictosweetorbittercultivarswere
in the sweet cultivar (Fig. 2, B and C). In the bitter also identified.Inallcultivarstested, PHwasfound to
almondcultivar,PHwasobservedintheapoplast(Fig. be localized in large aggregates or in vesicles. In the
2E).Tostudyinmoredetailthepossibledifferencesin sweetcultivarRamillete(Fig.4A),thelocalizationofPH
thelocalizationofPHinsweetandbittercultivars,the shifted from the symplast (i.e. 147, 154, and 168 JD) to
immunolocalizationofPHwasfollowedinthecourse the apoplast (182 and 220 JD) in the course of fruit
offruit development in the seedusingtwo sweetand development (Supplemental Videos S1 and S2). A par-
twobittercultivars. allel analysis of the sweet cultivar Lauranne at 147 JD
showed a dual localization: PH was found in the
apoplast surrounding some of the cotyledon cells but
ImmunolocalizationofPHinAlmondSeedsduringFruit
was found in the symplast of other cotyledon cells, as
Development in Sweet and Bitter Cultivars
visualized by the overlay images reflecting the super-
The localization of PH protein in different tissues position of the green color representing immuno-
(tegument, nucellus, endosperm, and embryo) of staining of PHs and the magenta color obtained by
1918 Plant Physiol. Vol. 158, 2012
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Prunasin Hydrolases in Sweet and Bitter Almonds
Figure2. Seedsfromtwoalmondcultivars,sweet(Lauranne;A–C)andbitter(S3067;D–F),at154JDwerecross-sectionedto
performimmunolocalizationstudiesusinga polyclonal antibodyagainstthesequenceINKKGIEYYpresentinthePHsfrom
almond (green vesicles). PHs were detected in the tegument (t), inner epidermis of the tegument (iet), nucellus (n), and
endosperm(en).Magentacolorrepresentsthecellwall(cw)andtheDNA/RNAstructuresstainedwithpropidiumiodideinthe
confocalstudies.ArrowsindicatethePHvesiclesinthecellsbelongingtotheinnerepidermisofthetegument.AandDshow
three-dimensionalpresentationsofthezstack,whereasB,C,E,andFpresentmagnificationsofthecellsintheinnertegument
withthesameviewanddifferentviewangles.A,B,D,andEshowviewsofopencells:inthesweetLaurannecultivar(AandB),
thecellwalloftheinnerepidermisofthetegumentremainsdevoidofPHvesicles,whereasinthebitterS3067cultivar(DandE),
PHvesiclesareobservedwithinthecellwall.[Seeonlinearticleforcolorversionofthisfigure.]
propidium iodide staining of cell walls and nuclei. At combinedtissuesofthenucellusandendosperm(asit
thesubsequentstagesoffruitdevelopmentinLauranne wasnotpossibletoseparatethesetissues,especiallyin
seeds,thePHswerefoundtobelocatedinthesymplast themidseason),andfromtheembryocomposedofthe
(154 and 168 JD), and in the final stages of fruit devel- two cotyledons and the small embryo axis. The coty-
opment, PH immunostaining was observed in the ledons are the final tissue where amygdalin is stored
apoplast (182 and 192 JD). In the bitter cultivars (Fig. (Frehner et al., 1990). Incubation of extracts from the
4B),thepatternofPHimmunostainingwasmoreorless tegument or from the combined nucellus and endo-
reversed compared with the observations with the spermwith20nmolofprunasinresultedintherelease
sweetcultivars.Intheearlytomiddevelopmentalstages of minute amounts of HCN, reflecting very low PH
oftheembryo,PHimmunostainingwasobservedinthe activity. In contrast, assays with the cotyledon extract
apoplastinthecotyledons(S3067at147JDandGorkiat released more than 50-fold higher amounts of HCN
147and154JD),evenfillingtheintercellularspaces(i.e. using the same amount of total protein from each
S3067at168JD).Atthelaterdevelopmentalstages,the tissue (Fig. 5A). The ability of the cotyledon prepara-
localizationofthePHsshiftedtothesymplast(S3067at tions to release HCN varied from one cultivar to the
220 JD) and to some extent the apoplast (Gorki at 192 other, although similarities were observed between
JD).Twovideosareprovided(SupplementalVideosS1 sweetandbitterphenotypes.Inthesweetcultivars,the
and S2) to more clearly document the symplastic or activityfluctuatedsignificantlythroughouttheseason,
apoplastic localization of PH. When the localization of whereas in general, the activity in the bitter cultivars
PH is in the symplast, a screen of the z-stack reveals decreased as the fruit matured. These differences
green-staining zones surrounded by a nonstaining re- could be due to fruit developmental differences or
gion representing the cell wall and apoplast space. At caused by the presence of multiple PH isoforms. In
developmental stages where PH is localized in the control experiments, a crude commercially available
apoplast, the stained patterns enclose nonstaining emulsin preparation from almonds (Sigma) was
domainsreflectingtheinternalcellularspace. added instead of an almond extract to liberate the
maximum amount of HCN (20 nmol). A negative
Biochemical Measurements of PH Activity during Fruit control,preparedwiththereactionbufferinsteadofan
Development in Almond Seeds almond extract, released about 0.5 nmol of HCN.
In parallel to the analysis of PH localizationduring
Detection of PH Isoforms in Almond Cotyledons by
seed development, the enzymatic PH activity was
SDS-PAGEfollowing Western Blotting and by Fast Blue
determinedbymeasuringtheabilityofcrudealmond
BB Salt Staining
extracts to catalyze the release of HCN following
incubationwithprunasin.Measurementswerecarried A single immunoreactive protein band with an
out using dialyzed extracts from the tegument, from apparent molecular mass of 63 kD was detected in
Plant Physiol. Vol. 158, 2012 1919
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Sa´nchez-Pe´rez et al.
Figure 3. Immunolocalization studies of prunasin localization in four different almond cultivars in the course of fruit
development.ThecultivarsRamillete(sweet),Lauranne(sweet),S3067(bitter),andGorki(bitter)wereanalyzedforPHcontent
attwodifferenttimepointsduringmidseason(154and182JD)offruitdevelopmentusingapolyclonalantibodyagainstthe
sequenceINKKGIEYYpresentinthePHs(greencolor)fromalmond.ThefirstfourcolumnsrepresentoverlayimagesofPH
detectedbythespecificantibody(green)andtranslucentlightmicrographstovisualizecellwalls(gray).Themarkedareaina
panelrepresentstheareashownathighermagnificationinthepaneltotherightinthesamerow,exceptforGorki,wherethe
sourceoftheimagesincolumnsdandearederivedfromthemarkedsegmentsincolumnc.Thelastcolumnrepresentsthree-
dimensionalimagesobtainedusingsectionscontainingmainlyendospermcellsstainedwithpropidiumiodideinmagentacolor
andgreenvesiclesthatrepresentthePHs.Abbreviationsareasfollows:endosperm(en),innerepidermisofthetegument(iet),
tegument(t),sometimeswithvascularbundles(v),andouterepidermisofthetegument(out).Inasmallnumberofthesections,
theembryo(em)orthenucellus(n)remainedattachedtotherestofthetissue.Inthelaterstageshown(182JD),thetegument
cellshavecollapsed.[Seeonlinearticleforcolorversionofthisfigure.]
all four cultivars following SDS-PAGE (Fig. 5B). b- possiblecomplexeswithotherproteinsnotdenatured
Glucosidasesretaintheircatalyticpropertiesfollowing bytheelectrophoreticconditionschosen.TheFastBlue
SDS-PAGE when the enzyme-containing samples are BBsalt-stainingmethodismoresensitivethanwestern
not boiled before application. Protein bands contain- blotting, explaining why these additional bands were
ing b-glucosidase activity, therefore, can be detected not observed on the immunoblot (Fig. 5B). When
followingincubationwiththeFastBlueBBsaltandthe these assays were performed to follow the profile of
6-bromo-2-naphthyl-b-D-glucopyranoside, a general b-glucosidase activity in the course of fruit develop-
substrate for b-glucosidases. Several Fast Blue BB- ment, all four cultivars showed a dominant immu-
generated reddish staining bands were obtained (Fig. nostainingbandwithanapparentmassof70kDwith
5C). The bands were excised from the gel and incu- PH activity and a number of weaker staining bands
bated with prunasin and amygdalin, as described forwhichtheintensityandmobilityvaried(Fig.5D).
previously, to analyze whether the b-glucosidase cat-
alyzed HCN release and thus represented prunasin
Isoforms of PH and Amygdalin Hydrolase in
and amygdalin hydrolases, respectively. Two protein
Almond Embryos as Analyzed by Liquid
bands with apparent masses of 70 and 150 kD con-
Chromatography-Tandem Mass Spectrometry
tained PH activity, while two bands with apparent
masses of 150 and approximately 190 kD showed A liquid chromatography-tandem mass spectrome-
amygdalinhydrolysisactivity.Thissuggests thepres- try(LC-MS/MS)approachwasundertakentoidentify
ence of homodimeric or heterodimeric protein com- thepresenceofPHhomologsinthetwosweetandtwo
plexes of prunasin and amygdalin hydrolases and bittercultivarsbasedonproteinextractsfromembryos
1920 Plant Physiol. Vol. 158, 2012
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Prunasin Hydrolases in Sweet and Bitter Almonds
Figure4. (Figurecontinuesonfollowingpage.)
Plant Physiol. Vol. 158, 2012 1921
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Sa´nchez-Pe´rez et al.
Figure4. (Legendappearsonfollowingpage.)
1922 Plant Physiol. Vol. 158, 2012
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
Prunasin Hydrolases in Sweet and Bitter Almonds
Figure 5. A, HCN released (nmol)
from the tegument and nucellus (teg
+nuc)andembryo(emb)intwosweet
cultivars (Ramillete [R] and Lauranne
[L])andtwobittercultivars(S3067[S]
andGorki[G])followingincubationof
crudeproteinextractswith20nmolof
prunasin at different stages of fruit
development (133–220 JD). B, Total
protein extracts were analyzed by
SDS-PAGE, stained with Coomassie
Brilliant Blue, and PHs present were
visualized by immunolocalizationus-
ing a polyclonal antibody against the
sequenceINKKGIEYYpresentinthePHs
fromalmond.C,Totalb-glucosidasecon-
tentwasassayedusingFastBlueBBsalt
stainingofSDS-PAGEfollowingtheap-
plication of crude protein extracts of
cotyledonsofthefourdifferentcultivars
harvested at 133 JD and revealed the
presenceofdifferentisoformsofPHs(PH)
andthepresenceofamygdalinhydrolase
(AH).D,Thesameassaywasusedto
monitor the presence of different b-
glucosidases during fruit develop-
mentinthefourcultivars.[Seeonline
articleforcolorversionofthisfigure.]
composedofthetwocotyledonsandthesmallembryo activity. Prunasin and amygdalin hydrolases have
axis isolated at 140 JD. At this developmental stage, expected masses of approximately 60 kD. Fast Blue
the cotyledon tissues are low in lipids but rich in BBstainingconfirmedthatb-glucosidaseactivitywas
protein, amygdalin has started to accumulate in the associated with proteins within this mass range. Gel
cotyledonsofthebittercultivars,andminuteamounts blocks covering the mass range 50 to 75 kD were ex-
ofprunasinarepresentinallfourcultivars.Thus,this cisedfromthegelstobeabletoidentifyb-glucosidase
developmentalstagewouldbeexpectedtobesuitable isoforms by digestion with trypsin. Tryptic peptides
for isolating enzymes involved in the breakdown or wereanalyzed by LC-MS/MS, and protein identifica-
turnover of cyanogenic glucosides in the sweet culti- tionwascarriedoutbycomparingtheMSandMS/MS
vars.Theproteinextractsofthefourdifferentcultivars spectra with the protein sequences in the National
weresubjectedtoSDS-PAGEandthegellanesstained Center for Biotechnology Information (NCBI) non-
witheitherCoomassieBrilliantBlueorwithFastBlue redundant protein sequence database. Preliminary
BB salt to identify proteins containing b-glucosidase ion-trap experiments identified several putative PH
Figure4. A,ImmunolocalizationstudiesofPHs(greencolor)intheembryooftwosweetgenotypes(RamilleteandLauranne)at
differenttimepointsduringfruitdevelopment(Ramillete,147,154,168,182,and220JD;Lauranne,147,154,168,182,and
192JD)usingapolyclonalantibodyagainstthesequenceINKKGIEYYpresentinthePHsfromalmond.ThepresenceofDNA/
RNAstructures(i.e.nucleus[n])andcellwall(cw)isvisualizedbypropidiumiodidestaining(magentacolor)andindicatedby
dottedarrows.ThelocalizationofthePHchangesduringfruitdevelopmentfrombeingmainlyinthesymplast(sy;i.e.Ramillete
at147,154,and168JDandLauranneat147,154,and168JD)tobeingpredominantlyfoundintheapoplast(ap;i.e.Ramillete
at 182 and 192 JD and Lauranne at 182 JD), as indicated by solid arrows, filling even the intercellular spaces (is). B,
ImmunolocalizationstudiesofPHs(greencolor)inthecotyledonoftwobittergenotypes(S3067andGorki)atdifferenttime
pointsduringfruit development (S3067,147,154,168,182,and220 JD; Gorki,147,154,168,182,and192JD) usinga
polyclonalantibodyagainstthesequenceINKKGIEYYpresentinthePHsfromalmond.ThepresenceofDNA/RNAstructures
(i.e.nucleus[n])andcellwall(cw)isvisualizedbypropidiumiodidestaining(magentacolor)andindicatedbydottedarrows.
ThelocalizationofthePHchangesduringfruitdevelopmentfrombeingmainlyintheapoplast(i.e.S3067at147,154,168,and
182JDandGorkiat147,154,168,and182JD),withoccasionalpresenceintheintercellularspaces(is),tobeingfoundinthe
symplast(i.e.S3067at220JDandGorkiat192JD).[Seeonlinearticleforcolorversionofthisfigure.]
Plant Physiol. Vol. 158, 2012 1923
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Sa´nchez-Pe´rez et al.
homologs to PHs of sweet cherry (Prunus avium) and identify all peptides present in the eluate from the
black cherry. Sequencing of the tryptic fragments HPLCcolumnbecauseoftherestrictionintimepoints
derived from the Coomassie Brilliant Blue-stained at which an MS/MS fragmentation may be recorded.
gel afforded 22 unique peptide sequences putatively Thus, peptides may be present in a sample even
derivedfromisoformsoffivePHsandoneisoformof though they were not registered by a sequence. To
amygdalin hydrolase, whereas analysis of the Fast test if this was indeed the case when PH homologs
Blue BB salt-stained gel afforded only nine unique were identified by the detection of different peptides
peptide sequences, demonstrating that SDS-PAGE is among the cultivars, ion chromatograms displaying
moresuitableforproteinidentificationbyLC-MS/MS the intensity of the differentially identified peptide
(data not shown). To obtain additional sequence in- mass-to-chargeratio (m/z)valuewereextracted.Inall
formation for the putative PH homologs, embryo samples, the peptide mass in question (within 62
extracts of the four almond cultivars were subjected ppm)elutedfromtheLCcolumnatthesameposition
to SDS-PAGE, stained with Coomassie Brilliant Blue, ofthegradient(610s).Thus,theidentifiedhomologs
and then processed as described above. The resulting are represented by the same peptides in all four
peptides were subjected to LC-MS/MS by the more cultivars; consequently, wecould notidentifycultivar-
sensitiveOrbitrapinstrument.Thesameprunasinand specific PH isoforms (Table I). Clearly, more peptides
amygdalin hydrolase homologs were identified as wereidentifiedderivedfromPHhomologs1,2,4,and
being present in all four cultivars. In most cases, the 5 from black cherry and b-glucosidase from sweet
homologswereidentifiedbythesamematchedtryptic cherry than from amygdalin hydrolase I and PH 3
peptides in all four cultivars, but in a few cases, a from black cherry. It is likely that the isoforms that
homologwasidentified by adifferentpeptidedepen- afforded more peptide hits are more similar to abun-
dent on the cultivars (Table I). LC-MS/MS does not dantisoform(s)inalmond.
TableI. Identifiedamygdalinhydrolase,PH,andb-glucosidasepeptidesequencesfromRamillete(R),Lauranne(L),S3067(S),andGorki(G)
cotyledons
Proteinsmigratingwithinthe50-to75-kDmassregionfollowingSDS-PAGEweredigestedwithtrypsinandanalyzedbyLC-MS/MS.Theresulting
pairs of peptide mass data and fragment ion (sequence) data were searched against the NCBI nonredundant protein database. X, Sequence is
identified(forthecriteriaforidentification,see“MaterialsandMethods”).(X),Notfragmentedbutpresentinextractedionchromatograms(m/z65
ppm)asadistinctchromatographicpeakelutingatthesametimeastheothersandwiththesameisotope(charge)distribution.
Genotype IsoformAssignment
PeptideSequence
Ra La Sa Ga AHIb PH1b PH2b PH3b PH4b PH5b PabGb
AASDWLYVYPK X X X X X X
AYADLCYK X X X X X
DMGLDAYR X X X X X X X X
EKYNDPIMYITENGMDEFNNPK X X X X X
FGINYIDYDNGLER X X X X X X
FGINYVDYDNGLKR X X X X X X X
FSISWSR X X X X X X X X X X
GDYPQSMR X X X X X X
GLYDLVLYTK X X X X X X X X
HWTTLNEPYTISNHGYTIGIHAPGR X X X X X X
ITDGSNGDVAIDQYHR X X X X X X X X
IVDDFK X X X X X X
KFGDR X X X X X
LPNFTEEQSK (X) (X) (X) X X X X X X
LSTHWFK X X X X X X X X
SFLKK X X X X X
SFLKR X X X X X
SIVGER X X X X X
SLSGSYDYIGVNYYSAR X X X X X
THFDTLFPGFTFGAATAAYQLEGAANIDGR (X) (X) X X X X
VKHWTTLNEPYTISNHGYTIGIHAPGR (X) X X (X) X X
YASAYPK X X X X X
YKEDVAIMK X X X X X X X X X
YNDPIMYITENGMDEFNNPK X X X X X X X
Hits 24 24 24 24 4 9 12 1 8 14 12
aPeptidesequencesobtainedfromRamillete,Lauranne,S3067,andGorkiandidentifiedbyNCBIsearchesaspartofamygdalinhydrolase,PH,or
b-glucosidases. bThepeptidesequencesarepartoftheproteinsequencesaccordingtoNCBI(cDNAaccessions):AHI(AF411130),amygdalin
hydrolasefromblackcherry;PH,PHisoformsfromblackcherryasfollows:PH1(U50201),PH2(AF221527),PH3(AF221526),PH4(AF411009),
PH5(AF411131);PabG,b-glucosidasefromsweetcherry(AAA91166).
1924 Plant Physiol. Vol. 158, 2012
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.