Plant Physiol. (1977) 59, 647-652

Subcellular Localization of the Cyanogenic Glucoside of Sorghumby Autoradiography1

Received for publication November 8, 1976 and in revised form December 6, 1976

JAMES A. SAUNDERS AND Epic E. CONNDepartment of Biochemistry and Biophysics

CHIN Ho LIN2 AND C. RALPH STOCKINGDepartment of Botany, University of California,

ABSTRACT

The use of microsutoradiography at the electron microscopic levelindicates that the vacuole is the site of accumulation of the cyanogenicglucoside ofSorghum bicolor. When a specific biosynthetic precuror ofdhurrin, p-hydroxy[3,5-3H]phenylacetaldoxime, was used, 90% of thetritium label was recovered in the vacuoles of tissue prepared for mi-croautoradiography. L-[3,5-3HJTyrosine and D_[1-3H(N)Jglucose, non-specific precursors of dhurrin, of differing solubilities and biosyntheticcapacity, were also fed to the shoots. The data obtained from thesecontrols indicated that the high recovery of label in the vacuole ofaldoxime-fed shoots was not indicative simply of the size of the vacuole,nor was it a result of movement of labeled compounds during prepara-tion of the tissue for electron microscopy. The problem of movement ofthese labeled compounds during dehydration of tissue was dramaticallyreduced by chemical dehydration in 2,2-dimethoxypropane in less than30 minutes rather than with routine dehydration in acetone or alcoholseries for 24 hours.

The technique of microautoradiography, although it shouldyield valuable information on the localization of water-solublenatural products, has not been extensively employed in plantsdue to the technical difficulties involved in the preservation ofthe tissue (18). This is particularly evident at the electron micro-scopic level where one must contend with stabilizing the move-ment of the water-soluble compounds in question and at thesame time assure adequate preservation of the tissue so as tomake the electron photomicrograph informative. The mostwidely used techniques to date have been those of freeze-substi-tution of the plant tissue in acetone or propylene oxide (13, 14,22) or the freeze-drying of the plant tissue followed by infiltra-tion with xylene and embedding in plastic or paraffin (15, 19). Inspecial cases in which the water-soluble compound can be pre-cipitated before dehydration, freezing is not necessary (19).The present study was undertaken to investigate the subcellu-

lar localization of the cyanogenic glucoside dhurrin in Sorghumseedlings by microautoradiography. As dhurrin and its metabolicprecursors are water-soluble (12), we employed a newly devel-oped technique for dehydration of plant tissue (Lin, Falk, Stock-ing, private communication). Using this technique, it was possi-

I This work was supported in part by National Science FoundationGrants BMS 72-02275-A02 to C. R. S., PCM 74-11997-AO1 to E. E.C., and United States Public Health Service Grant GM 05301-19 to E.E. C.

2 Present address: Botany Department, National Chung-Hsing Uni-versity, Taiwan.

Davis, California 95616

ble to fix and dehydrate shoot tissue in less than 1 hr, therebysignificantly reducing the likelihood of movement of the labeledcompounds. When a relatively specific precursor of dhurrin,tritium-labeledp-hydroxyphenylacetaldoxime, was administeredto Sorghum shoots, 90% of the silver grains were localized in thevacuoles.

MATERIALS AND METHODS

Plant Material. Fruits of Sorghum bicolor (Linn) Moench,variety Sordan 70, were obtained from Northrup King and Co.,Lubbock, Tex, and germinated in the dark at 25 C on cottongauze saturated with water. Two-day-old seedlings with a shootlength of 3 cm were excised 1 cm from the caryopsis underwater. The excised shoot was transferred to the appropriatelabeled compound in aqueous solution and incubated in the lightfor periods up to 8 hr.

Chemicals. The dehydrating agent, 2,2-dimethoxypropane,was obtained from Aldrich Chemical Co., D-[1-3H(N)]glucosefrom New England Nuclear, and L-[3,5-3HJtyrosine from Amer-sham/Searle. p-Hydroxy-[3,5-3H]phenylacetaldoxime was syn-thesized in approximately 20% yield from L-[3,5-3H]tyrosine bythe following sequence: 206 nmol of L-[3,5-3H]tyrosine, 485mCi/mmol) were oxidatively deaminated to p-hydroxyphenylpy-ruvic acid by the action of 1.08 mg L-amino-acid oxidase and1.11 mg catalase in 100 .lI 0.05 M K-phosphate (pH 7.3) duringa 20-min incubation at room temperature. The p-hydroxyphen-ylpyruvic acid was then converted to its oxime and the latter inturn reduced to N-hydroxytyrosine by the technique of Ahmad(1) as modified by Moller (in preparation). The N-hydroxytyro-sine was lyophilized to dryness and decarboxylated to p-hydrox-yphenylacetaldoxime by incubation at room temperature for 1 hrin 1 N NH40H. The aldoxime was purified by TLC on Silica GelIB-F thin layer plates in benzene-ethyl acetate (5:1).

All other chemicals used were of reagent grade or better.Cyanide was determined by hydrolysis of plant tissue with al-mond emulsin in center well flasks after homogenization withliquid N. The liberated HCN was trapped in 1 N NaOH andmeasured by the method of Epstein (11).

Incorporation of Labeled Compounds. The tritium-labeledcompounds were administrated to individual excised shoots byimmersing the cut surface in a 30-,ul volume. Time coursestudies indicated a maximum incorporation of tritium-labeledaldoxime into dhurrin after 8 hr of incubation, thus all scoring ofexposed silver grains was done on shoots fed for this interval oftime. In a typical experiment, the amounts of label fed per shootwere as follows: 25 ,uCi of -[1-3H(N)](glucose 18 Ci/mmol); 25,uCi of L-[3,5-3H]tyrosine (42 Ci/mmol); and 17 ,uCi of p-hydroxy-[3,5-3H]phenylacetaldoxime (485 mCi/mmol). Dhurrinwas identified in labeled shoots by grinding them in 70%

647

Plant Physiol. Vol. 59, 1977

4w

>~ ~ ~ ~ ~ ~ ~ ~~~ sg

'4'4;: 4#,~~. * P, 91 it-,' : r

FIG. 1. Primary leaf of 2-day-old dark-grown S. bicolor fixed with OS04 and dehydrated in 2,2-dimethoxypropane for 30 min. A: Representativecross-section showing the extensive vacuolation often seen in this tissue (x 4,800); B: adequate preservation of membrane and organelle structureare obtained using this method of tissue dehydration; (g), Golgi apparatus; (m), mitochondria; (er), endoplasmic reticulum; (n), nucleus; (p),plasmodesmata (x 19,000).

648 SAUNDERS ET AL.

LOCALIZATION OF CYANOGENIC GLUCOSIDES64

ethanol, removing the residue by centrifugation, and subjectingthe supernatant to TLC on Silica Gel TB-F layer plants. The

chromatograms were developed sequentially in 2-butanone-ace-

tone-water (15:5:3) and benzene-ethyl acetate (5:1), scanned

with a Packard radiochromatogram detector, and the dhurrin

and aldoxime spots eluted and counted in a Beckman scintilla-

tion counter. Conversion of aldoxime to dhurrin was calculated

both before and after the fixation and dehydration of the tissue.

Preparation of Tissue for Autoradiography. The apical (1 cm)of the excised shoot was sectioned into 2-mm segments and the

tissue fixed at 4 C in 2% 0S04 for min followed by three

washes for 3 min each in water. Dehydration was then accom-

plished by suspending the section in acidified 2,2-dimethoxypro-pane for 5 min five successive times (Lin, Falk, Stocking, private

communication). The tissue was then embedded in quarter-step

changes of Spurr's plastic medium (23), sectioned with a Serval

Porter-Blum microtome onto either copper or preferably gold

grids and poststained with 1% aqueous uranyl acetate followed

by lead citrate. The samples were then coated with Ilford L-4

liquid photographic emulsion by means of a looped copper wire

(5). After exposure times ranged from 2 weeks to 2 months, the

autoradiographs were developed in a 1/3 dilution of Microdol-X

and examined with a Zeiss EM-9A transmission electron micro-

scope.

Aliquots of all washing or treatment solutions were counted

on a Beckman scintillation counter equipped with an external

standard, to determine the extent to which labeled compoundswere removed during preparation of the tissue.

RESULTS

Although young Sorghum seedlings have long been known to

accumulate dhurrin (10), the specific localization of the cyano-

genic glucoside has not been determined. The 2-day-old, dark-

grown seedlings which have been used in biosynthetic studies (7)were chosen for their ease of manipulation and the extensive

synthesis and accumulation of dhurrin which occurs in the shoot.

The region of such seedlings above the apical node is approxi-

mately 1 cm in length and consists of a primary leaf surrounded

by a protective coleoptile coat; it is also extensively vacuolated,

particularly in those cells distant from the vascular regions (Fig.

A).The amount of dhurrin in the primary leaf (25-30 mg/g fresh

weight, Table I) does not differ significantly from that of the

coleoptile in the same region. However, it is one or more orders

of magnitude higher than the amounts found below the first

node, in the root, or in the caryopsis (Table I). There appears to

be a gradient of glucoside concentration away from the caryopsisin dark-grown tissue as the roots also contain higher levels than

found in the fruit.

When the amount of cyanogenic glucoside is expressed on a

dry weight basis, the concentration of dhurrin in the shoot

approaches 300 mg/g dry weight or approximately 30% of the

dry weight of the tissue. High levels of cyanogenic glycosides are

not restricted to young Sorghum seedlings, however, as Dement

and Mooney (9) have reported concentrations of prunasin as

high as 7% dry weight leaves of Heteromeles arbutifolia, an

evergreen shrub.

To investigate the subcellular localization of the water-soluble

cyanogenic glucoside in Sorghum seedlings by microautoradiog-raphy, it was necessary to minimize the movement of the labeled

compounds during fixation and dehydration procedures. For this

reason, a newly developed technique was used which permittedthe dehydration of plant tissue in less than 30 min (Lin, Falk,

Stocking, private communication). It consists of rapid fixation by0S04, dehydration by immersion of the tissue five successive

times for 5 min in acidified 2,2-dimethoxy-propane, and transfer

directly into Spurr's plastic (23), all within 1 hr. This rapidprocedure produced acceptable photomicrographs (Fig. 1, A

and B).

p-Hydroxyphenylacetaldoxime was chosen to label the cyano-

genic glucoside in intact Sorghum shoots because it is only three

reactions removed in the biosynthetic sequence for dhurrin (7).The aldoxime also has the advantage of not being metabolized to

other cellular constituents as are tyrosine or glucose, which were

also employed in this study.When p-hydroxy- [3 ,5-3H]phenylacetaldoxime was fed to

Sorghum shoots and the tissue subsequently fixed and dehy-drated, 26% of the radioactivity taken up initially by the shoots

was removed. Much of this loss from the tissue presumablyoccurred during the sectioning procedure necessary for proper

infiltration of the several reagents required. More important,however, the radioactivity which remained in the fixed and

dehydrated tissue was shown to reside almost exclusively in

dhurrin in the following manner. Tissue sections prepared for

embedding in plastic were instead homogenized and extracted in

70% ethanol. The extract was then centrifuged, concentrated,and examined by TLC for dhurrin. This procedure showed that

greater than 98% of the radioactivity in the ethanol extract was

contained in dhurrin. This figure is consistent with the demon-

strated effectiveness of p-hydroxyphenylacetaldoxime as a pre-

cursor of dhurrin (12) and suggests that the radioactivity re-

moved in fixation and dehydration could represent largely un-

metabolized aldoxime.

After exposure and development, the specimens were exam-

ined under a transmission electron microscope, and the location

of silver grains was scored on the basis of association with

specific cellular organelles. Figure 2A is a typical autoradiographprepared from shoots fed tritium-labeled aldoxime and magni-fied sufficiently not only to differentiate subcellular organelles

Table IOuantitative Determination of Dhurrin in Dark G-rown Sorghum

Two-day-old dark-grown seedlings consist of a 3-ca shoot and a 4-cm root system. TLhe region ofthe shoot above the apical node is approximately 1 cm in length and consists of a central primaryleaf surrounded by a protective coleoptile. Experiment A consists of 3 replicates of 20 seedlings.Experiment B consists of 3 replicates of 10 seedlings each such that the nodal region contained theapical node of each seedling.

Source froeshmwt D. mg Dhurrin/GFW S.D. % Dhurrin/GDW S.D.

Expt. A Coleoptile 93.7 (10.4) 29.2 ( 3.2) 30.9 ( 3.5)Leaf 89.8** ( 6.6) 27.9 ( 2.0) 29.7 ( 2.2)

Expt. B Apical 5 mm 74.3 (28.0) 23.1 ( 8.7) 24.6 ( 9.3)Nodal Region 7 mmn 26.6 (11.7) 8.3 ( 3.7) 8.8 ( 3.9)First Internode 12.8 ( 5.0) 4.0 ( 1.6) 4.2 ( 1.7)Caryopsis 0.15 (0.11) 0.05 (0.04) 0.01 (0.005)Root 14.2 (2.4) 4.4 (0.7) 7.2 C1.2)

*Stadad deviation.*"Not significantly different from Coleoptile at 0.01 probability level.

Plant Physiol. Vol. 59, 1977 649

Plant Physiol. Vol. 59, 1977

awa.

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i.a.If ;.

er

t44

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¼* .,

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FIG. 2. Microautoradiographs of shoot tissue from 2-day-old dark-grown S. bicolor which has been dehydrated in 2,2-dimethoxypropane andexposed under II L-4 emulsion at 4 C for 1 month. Preservation of tissue after development of the section was sufficient to identify and score silvergrains as to their location in six major organelles (Table II). A: p-hydroxy-[3,5-3H]Phenylacetaldoxime-fed shoot tissue during an 8-hr incubationperiod in which 90% of the recovered label was localized in vacuolar elements (x 15,000); B: L-[3,5-3H]tyrosine-fed shoot tissue during an 8-hrincubation period in which the recovery of the label was significantly different from the aldoxime-fed tissue but not significantly different fromtritium-labeled glucose-fed shoots; (v), vacuole; (p), plastid; (n), nucleus; silver grains indicated by arrows (x 11,000).

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650 SAUNDERS ET AL.

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LOCALIZATION OF CYANOGENIC GLUCOSIDES

but to identify the exposure tracks clearly. By scoring over 700grains in order to obtain a representative sample, we observedthat almost 90% of the tritium label was located in the vacuolarsystem of the cell (Table II). The remaining 10% of the silvergrains were distributed among the plastids, nuclei, cell walls, andelements of the cytosol not otherwise specifically mentioned. Itshould be noted that this undefined cytosol includes mitochon-dria, Golgi apparati, and other small organelles often obscuredby the size of the silver grain itself.

In several cases, vesicles of a small size were seen which mayhave represented either vacuolar precursors or (in some cases)artifacts of preparation such as swollen double membranes. Thepercentage of silver grains associated with these small vesicleswas always low. The number of grains associated with vesicularelements of probable vacuolar origin was included in Table II inthe total vacuolar system.As both dhurrin, the labeled products of the reaction sequence

and the aldoxime precursor are soluble in water, it was necessaryto consider the possibility of random movement of these labeledcompounds within the cell. Since the vacuole may often consti-tute as much as 90% of the volume of the cell (Fig. 1A),appropriate controls were required to establish that the distribu-tion of silver grains observed in Table II was not a value resultingprimarily from the solubility of the labeled compounds examinedand/or the vacuolar size.

This problem was approached by feeding two additional triti-ated precursors of dhurrin which have solubilities bracketingthose of the aldoxime or dhurrin, but equally important, areprecursors of other cellular metabolites as well. The precursorsused were L-[3,5-3H]tyrosine (solubility, 0.45 mg/l) and D-[1-3H(N)]glucose (solubility approaching 1000 g/l). In addition tolabeling dhurrin in the Sorghum shoots (12, 17), these com-pounds could be expected to label proteins, phenolic acids,flavonoids, lignin, other glucosides, and cell wall constituents.The localization of silver grains in seedlings fed tritium-labeled

tyrosine or glucose is shown in Tables III and IV, respectively.The distribution pattern is similar for both compounds but signif-icantly different from that in the seedlings fed the aldoxime.Only about one-third of the radioactivity was present in thevacuole of plants fed either tyrosine or glucose. A typical mi-

TABLE II

Autoradiographic Distribution of Label from Aldoxime Fed Sorghum

Location Grains % of Grains StandardScored Scored Deviation

Vacuole 624 86.8G,T 4.6Small Vesicle 12 2.3 2.7Both Vacuole and 636 891IG,T33

Small VesiclePlastid 5 08G,T 1.1Nucleus 4 0.5T 0.7Cell Wall 18 2.7G,T 2.2Undefined Cytosol 52 69 G,T 2.9

TOTAL 715 100.0

G = significantly different from glucose fed shoots at 0.01 probabilitylevel.

T = significantly different from tyrosine fed shoots at 0.01 probabilitylevel.

TABLE III

Autoradiographic Distribution of Label from Tyrosine Fed Sorghum

Grains % of Grains StandardLocation Scored Scored Deviation

Vacuole 450 34.1A 4.6Small Vesicle 61 4.7 3.1Both Vacuole and 511 38.8A 5.8

Small VesiclePlastid 121 9.1A 2.9Nucleus 101 7.5A,G 2.5Cell Wall 150 11.5A 4.9Undefined Cytosol 433 33.1A 4.3

TOTAL 1,316 100.0

A = significantly different from aldoxime fed shoots at 0.01 probabilitylevel.

G = significantly different from glucose fed shoots at 0.01 probabilitylevel.

TABLE IV

Autoradiographic Distribution of Label from Glucose Fed SorghumGrains % of Grains StandardScored Scored Deviation

Vacuole 202 31.2A 2.1Small Vesicle 17 2.7 1.0Both Vacuole and 219 33.9A 2.8

Small VesiclePlastid 71 10.9A 2.8Nucleus 15 2.2T 2.4Cell Wall 92 14.1A 2.2Undefined Cytosol 252 39.oA 2.7

TOTAL 649 100.0

A = significantly different from aldoxime fed shoots at 0.01 probabilitylevel.

T = significantly different from tyrosine fed shoots at 0.01 probabilitylevel.

croautoradiograph from shoot fed tritium-labeled tyrosine isshown in Figure 2B.

DISCUSSION

Two-day-old dark-grown Sorghum seedlings contain high lev-els of cyanogenic glucosides; the apical cm of the tissue wasapproximately 30% dhurrin/g dry weight (Table I). It is gener-ally assumed (8, 9), but by no means proven, that the cyanogenicglucosides are physically separated in the cells of cyanogenicplants from the degradative enzymes, 18-glucosidase and hydrox-ynitrile lyase, which catalyze their decomposition. The rapidrelease of HCN which occurs when cellular structure is disruptedis attributed to the glycosides being brought into contact withthese hydrolytic enzymes. Stafford (24) has speculated that theglycoside might be accumulated in the vacuole of plant cells.This seems reasonable in view of the high concentrations ofdhurrin in Sorghum seedlings and the extensive vacuolation inthe shoot tissue (Fig. 1 A). The suggestion is furtherstrengthened by the tentative localization of f8-glucosidase inregions of the plant cell other than the vacuole, i.e. the reticu-lated cytoplasm and peripheral cell wall region (3, 16, 21) andthe cytoplasm in general (2). In contrast, Matile has describedthe presence of several hydrolyases in the isolated vacuoles ofZea (20).The application of microautoradiography in the study of wa-

ter-soluble cellular components requires techniques designed tominimize the movement or loss of radioactive solutes during thepreparation of tissue. Fisher et al. (13, 14) successfully used amethod of freeze-substitution for preservation of tissue involvingwater-soluble betacyanins in the vacuoles of beets as a marker.This procedure required anhydrous solvents and rigorous condi-tions of low humidity as even traces of water gave poor preserva-tion of tissue.When the technique of freeze-substitution was applied in a

preliminary investigation to young Sorghum seedlings, the fixa-tion and preservation of tissue were extremely poor. Instead, weused a newly developed technique that accomplishes the dehy-dration of the plant tissue in less than 30 min (Lin, Falk,Stocking, private communication). This technique, in our hands,produced acceptable photomicrographs of the experimental tis-sue (Fig. 1) and retained a major fraction (- 75%) of theradioactivity taken up by the shoots.Knowledge of the biosynthetic pathway for dhurrin (6, 7)

dictated the choice of tritium-labeled p-hydroxyphenylacetal-doxime to label specifically the cyanogenic glucoside in thisstudy. The aldoxime is known to be rapidly incorporated into theglucoside which has a turnover rate of over 20 hr (4). The rapidrate of synthesis of a large amount of dhurrin and its accumula-tion in the apical cm of the shoot tip (Table I) of Sorghumseedlings provide a suitable experimental tissue. Tritium-labeledglucose and tyrosine appeared to be informative metabolic con-trols since they not only can serve as precursors of a variety ofother cellular constituents.The use of tritium-labeled glucose and tyrosine with their

Plant Physiol. Vol. 59, 1977 651

Plant Physiol. Vol. 59, 1977

different solubilities as precursors also provided effective con-trols on the movement of label during tissue preparation. Thepattern of distribution of silver grains from both of these precur-sors was similar yet highly significantly different from that inshoots fed the aldoxime. This indicated that the pattern obtainedin shoots fed the aldoxime was not an artifact of preparation butrather an accurate indication of accumulation of the cyanogenicglucoside in the vacuole. The data obtained indicated that 90%of the dhurrin in young Sorghum shoots is present in the vacuole(Table II and Fig. 2A). The pattern of distribution of silvergrains observed for a large population of cells will reflect thehighest concentration of radioactivity, presumably the site ofaccumulation. The data do not permit, nor do they preclude, theconclusion that the site of dhurrin synthesis is necessarily thevacuole itself.

Acknowledgment-The authors acknowledge gratefully B. L. Moller for his help in thepreparation and purification of p-hydroxy-[3 ,5-3Hlphenylacetaldoxime.

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