Plant Physiol.
(1992) 98, 1423-1428
0032-0889/92/98/1 423/06/$01 .00/0
Development of the Potential for Cyanogenesis in Maturing
Black Cherry (Prunus serotina Ehrh.) Fruits1
Elisabeth Swain, Chun Ping Li, and Jonathan E. Poulton*
Department of Botany, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
Biochemical changes related to cyanogenesis (hydrogen cya-
nide 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 amyg-
dalin levels, and (c) levels of the catabolic enzymes amygdalin
hydrolase, prunasin hydrolase, and mandelonitrile lyase. During
phase 1 (0-28 days after flowering [DAF]), immature fruits accu-
mulated prunasin (mean: 3 micromoles/fruit) but were acyano-
genic because they lacked the above enzymes. Concomitant with
cotyledon development during mid-phase 11, the seeds began
accumulating both amygdalin (mean: 3 micromoles/seed) and the
catabolic enzymes and were highly cyanogenic upon tissue dis-
ruption. Meanwhile, prunasin levels rapidly declined and were
negligible by maturity. During phases 11(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 de-
velopmental stages.
Over 2650 plant species distributed throughout 130 families
of angiosperms, gymnosperms, and ferns release large quan-
tities of HCN2 upon tissue disruption or infection (22). Most
frequently, cyanogenesis (HCN production) arises during the
degradation of cyanogenic glycosides (19). Because tissue
maceration is required for large-scale cyanoglycoside catabo-
lism, it is generally assumed that these glycosides and their
respective catabolic enzymes are compartmentalized in intact
plants at either tissue or subcellular levels (18). Such com-
partmentation of the components constituting the "cyanide
bomb" prevents any premature HCN release until tissue
disruption allows their interaction. Given the well-docu-
mented toxicity of HCN (17), a role for cyanogenesis in plant
protection against herbivores, pathogens, and competitors is
appealing and has received some experimental support (8,
16). However, one should keep in mind the possibility that
the carbonyl compounds released during cyanoglycoside ca-
tabolism may also act as feeding deterrents (8). Recent studies
with germinating seedlings have shown that cyanogenic gly-
cosides and the related cyanolipids may additionally serve as
nitrogen storage compounds (24, 25).
' Supported by National Science Foundation grant DCB 89-17176.
2Abbreviations: HCN, hydrogen cyanide; AH, amygdalin hydro-
lase; PH, prunasin hydrolase; MDL, mandelonitrile lyase; TBS, Tris-
buffered saline.
1423
Received for publication August 9, 1991
Accepted December 2, 1991
The biochemistry of cyanogenic glycoside accumulation by
maturing seeds and fruits has so far received little attention
(4, 5, 7, 13-15). Cyanoglycoside levels have been monitored
during fruit development but, without exception, they were
not correlated with changes in levels of enzymes involved in
glycoside metabolism. The highly cyanogenic nature of rosa-
ceous stone fruits (e.g. almonds, peaches, cherries) has long
been known. Although the fleshy portions of these fruits are
traditionally regarded as acyanogenic and therefore innocu-
ous, the seeds, which accumulate the cyanogenic disaccharide
(R)-amygdalin, have been responsible for numerous cases of
acute cyanide poisoning of humans and domesticated and
wild animals (17). For several years, our laboratory has been
investigating amygdalin metabolism in black cherry seeds (10,
11, 30). Upon crushing mature seeds, amygdalin is rapidly
catabolized to HCN, benzaldehyde, and glucose through the
sequential action of the enzymes AH, PH, and MDL. To
provide further insight into the possible physiological roles
played by cyanogenic glycosides, we have followed the devel-
opment of the potential for cyanogenesis in maturing black
cherry fruits. Levels of the cyanogenic glycosides amygdalin
and prunasin and of the enzymes AH, PH, and MDL were
monitored from shortly after flowering until maturity. Wher-
ever possible, analysis was performed on separated fruit tissues
to provide greater insight into developmental processes.
MATERIALS AND METHODS
Chemicals and Chromatographic Materials
(R)-Amygdalin, (R)-prunasin, polyvinylpolypyrrolidone,
protease inhibitors, almond emulsin (type G-0395), horse-
radish peroxidase-conjugated goat anti-rabbit immunoglobu-
lin G, and glucose oxidase-peroxidase reagents were purchased
from Sigma Chemical Co. (St. Louis, MO). Chromatographic
materials were purchased from the following sources: Sepha-
dex G-25, Pharmacia Fine Chemicals (Piscataway, NJ);
DEAE-cellulose, Whatman Ltd. (Kent, UK).
Plant Materials
A single black cherry (Prunus serotina Ehrh.) tree, located
at Hickory Hill Park, Iowa City, was selected as source of
plant materials. Full flowering occurred on May 21, 1990.
Beginning 14 DAF and continuing at 7-d intervals until full
maturity, developing fruits were collected between 9:00 and
10:00 AM. Samples were enclosed in plastic bags and kept on
ice during transfer to the laboratory, where they were analyzed
1 424 SWAIN ET AL.
without delay for fresh and dry weights and for glycoside and
enzyme levels.
Analysis of Fresh and Dry Weights
On each sampling date, the fresh weight was determined by
counting and weighing a representative sample of fruits im-
mediately upon their arrival in the laboratory. For dry weight
analysis, a known number of cherries was weighed before
being dried at 80C until constant weight was attained. After
endocarp development began (first noted 28 DAF), cherries
were sliced in half with a razor blade to facilitate drying.
Analysis of AH, PH, and MDL Levels by Enzymatic Assay
At early developmental stages (0-21 DAF), enzyme levels
were determined in extracts derived from whole fruits as
described below. After endocarp development began (28
DAF), fruits were dissected before homogenization into two
fractions, namely the developing seeds (i.e. the contents of
the endocarp) and the remaining tissues (the pericarp), which
were processed separately. Plant tissue (4 g, derived from a
known number of cherries) was homogenized at 4C in a
mortar with 0.2 g polyvinylpolypyrrolidone, 1 g sand, and 20
mL of 0.1 M histidine-HCl buffer, pH 6.0, containing 1 mM
PMSF, 5 mM EDTA, and 1 ,uM tosyl-lysine chloromethylke-
tone. The homogenate was centrifuged at 17,600g for 25 min,
and the pellet was discarded. An aliquot (7-20 mL) of the
supernatant liquid was chromatographed on a Sephadex G-
25 column (40 x 2.5 cm) that was preequilibrated and eluted
with 20 mm sodium acetate buffer, pH 5.0. Fractions contain-
ing protein (monitored at 280 nm by an Isco V4 absorbance
detector) were pooled and used for estimation of PH, MDL,
and protein levels. For analysis of AH levels, AH and PH
were resolved by applying an aliquot (4 mL) of the G-25
eluate to a DEAE-cellulose column (2.5 x 1 cm) that had
been preequilibrated with 20 mm sodium acetate buffer, pH
5.0. AH was not retained by that matrix and was eluted by
washing the column with 20 mL of this buffer (11). To
confirm that ion-exchange chromatography had successfully
removed PH, the eluate was routinely assayed for PH activity.
,3-Glucosidase activity toward amygdalin (AH activity) and
prunasin (PH activity) was assayed in triplicate as described
by Kuroki and Poulton (10, 1 1). MDL activity was assayed
as previously described (30). Values shown represent the mean
of all trials.
Analysis of AH, PH, and MDL Levels by Immunoblotting
Crude seed homogenates containing AH, PH, and MDL
were prepared as described above and, after 1:1 dilution with
57.5 mM Tris-acetate, pH 4.8, containing 40% (v/v) glycerol,
were stored at -20C until processed. Equal volumes were
subjected to SDS-PAGE on 10% gels (21). Proteins were
electroblotted at 23C for 2 h onto nitrocellulose filters in 25
mM Tris, 200 mm glycine, 20% (v/v) methanol transfer buffer
using a Trans-Blot apparatus (Bio-Rad) at 60 V. After rinsing
the blot three times (10 min each) with TBS (10 mm Tris-
HCI, pH 7.4, containing 0.9% [w/v] NaCl), nonspecific pro-
tein binding sites were blocked by incubating filters for 30
Plant Physiol. Vol. 98, 1992
min at 23C with blocking buffer (5% [w/v] nonfat dry milk
powder and 0.05% [v/v] Tween 20 in TBS). The blot was
then transferred to fresh blocking buffer for another 90 min
before overnight incubation with primary antibody (for origin
and dilutions, see below). Unbound primary antibodies were
removed by three washes (10 min each) with blocking buffer.
The filters were incubated with horseradish peroxidase-con-
jugated goat anti-rabbit immunoglobulin G (diluted 1:3000
with blocking buffer) for 1 to 4 h at 23C followed by three
washes (10 min each) with TBS. The locations of antigen-
antibody complexes were visualized by incubating with 0.05%
(w/v) 4-chloro-1-naphthol and 0.05% (v/v) H202 in TBS
containing 16.7% (v/v) methanol.
Polyclonal antibodies were raised in New Zealand White
rabbits against trifluoromethanesulfonic acid deglycosylated
AH, PH, and MDL (27) and purified by affinity chromatog-
raphy where necessary. Their monospecificity was confirmed
by western immunoblotting analysis after resolving total seed
proteins by one-dimensional and two-dimensional SDS-
PAGE (for anti-MDL antibodies, see ref. 29; for anti-AH and
anti-PH antibodies, data to be published elsewhere). Before
use, anti-AH, -PH, and -MDL antibodies were diluted 1:500,
1:500, and 1:1000, respectively, with blocking buffer.
Protein Estimation
Protein content was estimated by the Bradford procedure
(2) with BSA serving as standard.
Analysis of Cyanogen Content
Prunasin and amygdalin were extracted from developing
fruits in methanol, resolved by HPLC, and quantitated by
assaying HCN levels after their complete enzymic hydrolysis.
A known number of cherries (or selected parts thereof) was
weighed, pulverized in liquid N2 in a mortar (precooled by
liquid N2), and extracted with 70 mL boiling methanol for 5
min. The sediment was recovered by filtration through glass
wool and reextracted with 50 mL boiling 70% methanol for
5 min. Insoluble components were removed by filtration, and
the combined filtrates were centrifuged at 17,600g for 10 min.
The supernatant was reduced in volume to 0.5 to 1.0 mL by
evaporation overnight under a fume hood followed by rotary
evaporation (below 50C). Cyanoglycosides were quantita-
tively transferred to 2-mL graduated tubes using 70% (v/v)
methanol as solvent, centrifuged in closed Eppendorf tubes
in a Beckman Microfuge E for 5 min, and stored at -20C
until analyzed. HPLC was performed using a Beckman 1 OA
pump linked to a Beckman 153 UV detector (254 nm).
Cyanoglycosides were resolved by injecting an aliquot (100
,uL) onto an Ultrasphere-ODS column (10 x 250 mm, Altex)
and eluting isocratically at room temperature with 38% (v/v)
methanol at 2.0 mL/min. Average retention times for amyg-
dalin and prunasin were 9.5 and 13.1 min, respectively.
Fractions were collected between 6 and 17 min, reduced to
dryness by rotary evaporation (under 50C), and redissolved
in 2 mL 0.1 M potassium phosphate buffer, pH 6.8. An aliquot
(0.5 mL) was incubated overnight at 37C in shaking Thun-
berg tubes with 0.3 mL almond emulsin (containing 7.2 units
,B-glucosidase activity). Liberated HCN was absorbed in traps
 CYANOGENESIS IN MATURING PRUNUS SEROTINA FRUITS 1425

containing 0.7 mL 1 M NaOH. The traps were rinsed with an

additional 0.2 mL 1 M NaOH, and the released HCN was A
measured by standard methods (1) using standard sodium
cyanide solutions for quantitation. When subjected to HPLC
and cyanide analysis as described above, recoveries of authen-
tic prunasin and amygdalin routinely exceeded 97%.
After endocarp development began (28 DAF), fruits were
dissected into two fractions, namely the developing seeds
(contents of the endocarp) and the remaining tissues (the
pericarp) for separate analysis. B
Presentation of Data
In keeping with earlier studies (7), the potential cyanogen-
icity of fruits during their maturation has been emphasized
here by expressing cyanogen and enzyme levels on a per fruit
(or fruit part) basis. To facilitate comparison with immuno-
blot analysis data, the enzyme levels are also given on a per g
fresh weight basis. The complexity of chromatographic tech- C
niques rendered unfeasible the processing of multiple samples
at each time-point. Nevertheless, each data point represents
an average derived from many (n = 8-750) fruits or fruit
parts.

RESULTS
Developmental Stages of Fruit Maturation D
In common with many other fleshy drupaceous fruits (26,
28), the development of P. serotina fruits showed three distinct
growth phases (12). During phase I (0-28 DAF), the fruits
exhibited a dramatic increase both in size and in fresh and
dry weights (Fig. 1). During mid-phase I, the endocarp began
differentiation and by 28 DAF, was quite woody and enclosed

Figure 2. Anatomy of black cherry fruits during three characteristic

phases of development. A, Mid-phase I. Easily visible are the nucellar
(n) and endosperm (e) tissues enclosed within the increasingly woody
endocarp (w). The embryo is difficult to detect at this stage. B, Mid-
phase 11. The cotyledons (c) of the rapidly developing embryo are now
clearly visible. C, Late phase II. The nucellar and endosperm tissues
400- III
diminish markedly in size as they supply nutrients for embryo devel-
opment. D, Phase Ill. The cotyledons now occupy the majority of the
seed volume, whereas the residual endosperm tissue is restricted to
300- two thin strips of cells associated with the testa. Ripening of the
mesocarp (m) includes an increase in cell size and accumulation of
anthocyanins and sugars.
200

glassy-looking nucellar and endosperm tissues (Fig. 2A).

100 Thereafter, a period of retarded growth (phase II; 29-65 DAF)
ensued during which the embryo developed within and at the
expense of the nucellus and endosperm (Fig. 2, B and C).
Cotyledons became visible to the naked eye approximately 42
0 20 40 60 80 DAF and, by the end of phase II (65 DAF), they occupied
most of the seed volume, whereas the residual endosperm
tissue was restricted to two thin strips of cells associated with
DAF the surrounding testa. At this time, the fruits began to redden
Figure 1. Growth of maturing black cherry fruits from flowering to as anthocyanins were produced. The last phase of fruit mat-
maturity. Mean fresh weights (0) and dry weights (A) were determined uration (phase III; 66-81 DAF) was characterized by a marked
as described in "Materials and Methods." increase in fresh and dry weights as the mesocarp ripened and
1 426 SWAIN ET AL.
the cherries finally assumed a black hue (Fig. 2D). Although
the lengths of phases I through III are sensitive to climatic
conditions and thus vary somewhat from season to season,
changes in levels of the cyanogens (and their catabolic en-
zymes) follow similar trends as those detailed below for the
1990 season.
Cyanogenic Glycoside Levels during Fruit Maturation
The levels of prunasin and amygdalin in whole fruits fol-
lowed markedly different time-courses during fruit matura-
tion (Fig. 3A). Paralleling observed changes in fruit weights,
prunasin levels rose quickly during phase I and reached
approximately 3.1 gmol/cherry by 28 DAF. At this time, the
seed and pericarp tissues contained roughly equal amounts of
prunasin (Fig. 3, B and C). After attaining a maximum of 3.5
Aumol/fruit at 42 DAF, whole fruit prunasin levels declined
throughout the remainder of phases II and III (Fig. 3A). In
contrast, amygdalin levels rose steeply from near zero at 35
DAF to exceed 5 ,mol/fruit by 76 DAF.
Beginning 28 DAF, fruits were also dissected into two
6- A (6). Direct enzyme assays provided an explanation for this
5
3-
=L 2-
4-B
4- B
C.0 3 antibody preparations having demonstrated monospecificities
0O 2-
0
lo 30 50 70
DAF
Figure 3. Levels of cyanogenic glycosides in whole fruits (A), seeds
(B), and pericarps (C) during fruit maturation. Prunasin (O) and amyg-
dalin (O) were extracted from pulverized tissues (n = 1 1-1 00, de-
pending on developmental stage) in boiling 70% methanol, resolved
by HPLC, and quantitated after enzymic hydrolysis. Each data point
represents the mean of two replicates whose variability did not
exceed the dimensions of the symbol shown.
Plant Physiol. Vol. 98, 1992
fractions, namely the developing seeds and the pericarp, for
individual analysis of their cyanoglycoside contents. Seed
tissues at 28 DAF, which consisted largely of nucellar and
endosperm tissues enclosed within the integuments, possessed
1.36 ,umol prunasin/fruit unit but lacked amygdalin (Fig. 3B).
Concomitant with cotyledon development (approximately 42
DAF), amygdalin levels increased rapidly and eventually ex-
ceeded 3 ,umol/seed by 76 DAF. In contrast, prunasin levels
declined during cotyledon development and were negligible
by 76 DAF. Similar changes in cyanogenic glycoside levels
occurred in the pericarp (Fig. 3C). Between 28 and 56 DAF,
prunasin levels remained roughly constant at approximately
1.6 ,umol/fruit unit before decreasing to 0.5 ,umol/unit by 76
DAF. Amygdalin began accumulating in the pericarp 35 to
42 DAF and steadily increased, reaching a plateau concentra-
tion of approximately 2 ,umol/unit at 76 DAF. At maturity,
cyanoglycosides were detectable in both exocarp (epidermis)
and mesocarp using the semi-quantitative Feigl-Anger test (6).
Levels of AH, PH, and MDL during Fruit Maturation
The ability of developing black cherry fruits to release
significant levels of HCN upon tissue disruption was first
noted during mid-phase II, as judged by the Feigl-Anger test
finding. Coincident with cotyledon development and amyg-
dalin accumulation, the levels of AH, PH, and MDL increased
rapidly and specifically within seed tissues (Fig. 4A). Such
dramatic increases in enzyme activity might result from (a)
enzyme induction, (b) activation of preexisting, inactive en-
zyme forms, or (c) loss of hypothetical inhibitors that sup-
pressed these activities during phase I. To distinguish between
these possibilities, proteins contained within cell-free extracts
of maturing seeds (28-81 DAF) were resolved by SDS-PAGE,
blotted onto nitrocellulose, and probed using three polyclonal
for AH, PH, and MDL, respectively. Immunoblotting re-
vealed that these proteins were absent from developing seeds
until their coordinate induction during mid-phase II (Fig. 4B).
First detected 49 DAF, their levels quickly increased, eliciting
strong immunostaining responses until maturity.
In contrast with their high activities in maturing seeds, AH,
PH, and MDL were not detectable in cell-free extracts of the
pericarp at any developmental stage by either direct enzyme
assay or immunoblotting (data not shown). Thus, despite its
high cyanoglycoside content during phases I through III (Fig.
3C), the pericarp appears acyanogenic because it lacks the
enzymic machinery to release HCN upon tissue disruption.
These data explain why macerated tissues from immature
fruits elicited only a weak response in the Feigl-Anger test
until exogenous ,B-glucosidases (almond emulsin) were pro-
vided (20). Such behavior is also exhibited by the mesocarp
of ripe, commercially marketed peaches (data not shown).
DISCUSSION
The pattern of accumulation of amygdalin and prunasin in
developing black cherries was similar to those seen in other
rosaceous stone fruits (4, 7, 14). Although prunasin predom-
inates during phase I, the synthesis of amygdalin during later
 CYANOGENESIS IN MATURING PRUNUS SEROTINA FRUITS 1 427

phases, concomitant with a decline in prunasin levels, causes

A. the disaccharide to predominate (>85%) at maturity. By
assaying the levels of AH, PH, and MDL in various fruit
parts, the current study reveals their tissue localization with
respect to those of the glycosidic substrates. It also identifies
r 4o
the developmental stages when fruits have the ability to release
;O s
HCN.
-
-,I) Z; -L.
Until mid-phase II, fruits are acyanogenic because they
P11
.0 =
E contain cyanoglycosides (mostly prunasin) but lack the cata-
In .!- bolic enzymes. Thereafter, the developing P. serotina seeds
i. --I
H accumulate both components of the so-called "cyanide bomb"
and are thus potentially highly cyanogenic. The mode of
K
cellular or subcellular compartmentation within uncrushed
MDL *A SO .- zi seeds that prevents the premature, suicidal degradation of the
-,;Z
N
..o
-1 .= cyanoglycosides is currently under investigation in our labo-
= -!!7
.4(l Q. ;: ratory using immunocytochemical techniques (29). Upon
n- tissue disruption, the glycosides are rapidly degraded by AH,
PH, and MDL releasing HCN and benzaldehyde (10, 11, 30).
f,
The documented toxicity of P. serotina toward livestock and
humans supports the notion that these reaction products serve
to defend the propagules against herbivory (9). Whether amyg-
40
dalin also serves as a nitrogen source for germinating seedlings
remains to be evaluated.
The flesh of rosaceous stone fruits such as peaches and
apricots is generally regarded as acyanogenic. Likewise, the
.2' 4('
fut
s ll black cherry pericarp is also acyanogenic. Although it accu-
DA

mulates high levels of cyanogenic glycosides throughout fruit

maturation, it lacks the enzymes AH, PH, and MDL that
B. would release HCN. This situation is not unique to Prunus
species. For example, few Australian Acacia species actually
AH possess the f3-glucosidases required to hydrolyze endogenous
s. . ; ,
cyanogenic glycosides (23). The physiological role(s) played
by the P. serotina pericarp glycosides is (are) unclear. It is
possible that the unhydrolyzed cyanoglycosides might con-
tribute toward defense through their bitterness, as was shown
for the glycoside cardiospermin (3). Certainly, immature fruits
PH suffer little predation until late-phase III, when the fruits
soften, change color, and become attractive to birds, notably
robins. At this time, the bitterness of the glycosides might be
offset by sugar accumulation. Pericarp cyanoglycosides could
also contribute to defense against herbivores through release
MDL of HCN and benzaldehyde if fruits were sufficiently damaged
as to allow mixing of macerated pericarp tissues with the seed
* - m m - - fl-glucosidases.
21 28 35 42 49 56 63 70 76 81 ACKNOWLEDGMENTS
DAF The authors wish to thank Mark Woerner for photographic repro-
Figure 4. Levels of AH, PH, and MDL in developing P. serotina ductions and Linda Donohoe and Tony Nevshemal for assistance in
seeds. A, Extracts of developing seeds (n = 8-750, depending on manuscript preparation.
developmental stage) were partially purified as described in "Materials
and Methods." Enzyme activities are expressed on a per fruit (0) or
a per g fresh weight (0) basis. Each data point represents the mean LITERATURE CITED
of three replicates whose variability did not exceed the dimensions 1. Aldridge WN (1944) A new method for the estimation of micro-
of the symbol shown. B, Immunoblot analysis of AH, PH, and MDL quantities of cyanide and thiocyanate. Analyst 69: 262-265
levels. Equal volumes (30 gL) of crude seed homogenates, derived 2. Bradford M (1976) A rapid and sensitive method for the quan-
from maturing seeds as described in "Material and Methods," were titation of microgram quantities of protein utilizing the prin-
subjected to SDS-PAGE and immunoblot analysis using polyclonal ciple of protein-dye binding. Anal Biochem 72: 248-254
antibodies monospecific for each protein. 3. Braekman JD, Daloze D, Pasteels JM (1982) Cyanogenic and
other glucosides in a neo-Guinean bug Leptocoris isolata.
Biochem Syst Ecol 10: 355-364
1 428 SWAIN ET AL.
4. Brink KR (1990) Accumulation of the cyanogenic glycosides
prunasin and amygdalin in apricot fruits. MS thesis. University
of California, Davis.
5. Dement WA, Mooney HA (1974) Seasonal variation in the 19.
production of tannins and cyanogenic glucosides in the cha-
parral shrub, Heteromeles arbutifolia. Oecologia 15: 65-76 20.
6. Feigl G, Anger V (1968) Replacement of benzidine by copper
ethylacetoacetate and tetra base as spot-test reagent for hydro-
gen cyanide and cyanogen. Analyst 91: 282-284 21.
7. Frehner M, Scalet M, Conn EE (1990) Pattern of the cyanide-
potential in developing fruits. Plant Physiol 94: 28-34
8. Jones DA (1988) Cyanogenesis in animal-plant interactions. In
D Evered, S Harnett, eds, Cyanide Compounds in Biology,
Ciba Foundation Symposium No 140. John Wiley & Sons, 22.
Chichester, UK, pp 151-176
9. Kingsbury JM (1964) Poisonous Plants of the United States and
Canada. Prentice Hall, Englewood Cliffs, NJ, pp 365-367
10. Kuroki GW, Poulton JE (1986) Comparison of kinetic and 23.
molecular properties of two forms of amygdalin hydrolase from
black cherry (Prunus serotina Ehrh.) seeds. Arch Biochem
Biophys 247: 433-439
1 1. Kuroki GW, Poulton JE (1987) Isolation and characterization of 24.
multiple forms of prunasin hydrolase from black cherry (Pru-
nus serotina Ehrh.) seeds. Arch Biochem Biophys 255: 19-26
12. Labrecque M, Barabi D (1984) Developpement du fruit de 25.
Prunus serotina (Rosaceae). Can J Bot 62: 195-206
13. Majak W, McDiarmid RE, Hall JW (1981) The cyanide poten-
tial of saskatoon serviceberry (Amelanchier alnifolia) and cho- 26.
kecherry (Prunus virginiana). Can J Anim Sci 61: 681-686
14. Nahrstedt A (1971) Die cyanogenen glykoside von Prunus avium 27.
L. und ihre verteilung wahrend einer vegetationsperiode. Dis-
sertation, Freiburg University 28.
15. Nahrstedt A (1973) Cyanogenesis in Cotoneaster-Arten. Phyto-
chemistry 12: 1539-1542
16. Nahrstedt A (1985) Cyanogenic compounds as protecting agents 29.
for organisms. Plant Syst Evol 150: 35-47
17. Poulton JE (1983) Cyanogenic compounds in plants and their
toxic effects. In RF Keeler, AT Tu, eds, Handbook of Natural 30.
Toxins, Vol 1, Plant and Fungal Toxins. Marcel Dekker, New
York, pp 117-157
18. Poulton JE (1988) Localization and catabolism of cyanogenic
Plant Physiol. Vol. 98, 1992
glycosides. In D Evered, S Harnett, eds, Cyanide Compounds
in Biology, Ciba Foundation Symposium No. 140. John Wiley
& Sons, Chichester, UK, pp 67-91
Poulton JE (1990) Cyanogenesis in plants. Plant Physiol 94:
40 1-405
Poulton JE, Shin SI (1983) Prunasin biosynthesis in cell-free
extracts from black cherry (Prunus serotina Ehrh.) fruits and
leaves. Z Naturforsch 38c: 369-374
Sambrook J, Fritch EF, Maniatis T (1989) Detection and analy-
sis of proteins expressed from cloned genes. In C Nolan, ed,
Molecular Cloning: A Laboratory Manual, Book 3. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp
18.47-18.54
Seigler DS (1991) Cyanide and cyanogenic glycosides. In GA
Rosenthal, MR Berenbaum, eds, Herbivores: Their Interaction
with Secondary Plant Metabolites. Academic Press, New York,
pp 35-77
Seigler DS, Maslin BR, Conn EE (1989) Cyanogenesis in the
Leguminosae. In CH Stirton, JL Zarucchi. eds, Advances in
Legume Biology. Monogr Syst Bot Missouri Bot Gard Vol 29,
Missouri Botanical Garden, St. Louis, pp 645-672
Selmar D, Lieberei R, Biehl B (1988) Mobilization and utiliza-
tion of cyanogenic glycosides. The linustatin pathway. Plant
Physiol 86: 711-716
Selmar D, Grocholewski S Seigler DS (1990) Cyanogenic lipids:
utilization during seedling development of Ungnadia speciosa.
Plant Physiol 93: 631-636
Sharma AI (1981) Studies on fruit development of peach (Prunus
persica cultivar 16-33). Prog Hortic 12: 23-30
Sojar HT, Bahl OP (1987) A chemical method for the deglyco-
sylation of proteins. Arch Biochem Biophys 259: 52-57
Tukey HB (1935) Growth of the embryo, seed and pericarp of
the sour cherry (Prunus cerasus) in relation to season of fruit
ripening. Proc Am Soc Hortic Sci 31: 125-144
Wu HC, Poulton JE (1991) Immunocytochemical localization
of mandelonitrile lyase in mature black cherry (Prunus serotina
Ehrh.) seeds. Plant Physiol 96: 1329-1337
Yemm RS, Poulton JE (1986) Isolation and characterization of
multiple forms of mandelonitrile lyase from mature black
cherry (Prunus serotina Ehrh.) seeds. Arch Biochem Biophys
247: 440-445