Plant Physiol. (1981) 68, 53-580032-0889/81/68/0053/06/$00.50/0

Wound-Induced Membrane Lipid Breakdown in Potato Tuber'Received for publication July 2, 1980 and in revised form December 18, 1980

ATHANASIOS THEOLOGIS' AND GEORGE G. LATIESBiology Department and Molecular Biology Institute, University of California, Los Angeles, California 90024

ABSTRACT

Freshly cut slices of potato tuber show an extensive loss of membraneUpid components which may be as great as 35% for phospholipids and 30%for glycolipids, in less than 15 minutes at 3 C. Phosphatidyl-choline,phosphatidyl-ethanolamine and mono- and di-galactosyl diglycerides com-prise the bulk of the lipids that are degraded. Concomitantly, there is apronounced loss of linoleic and linolenic acids. Whereas degradative eventselicited by slicing proceed to a depth of at least 10 millimeters from thesurface, phospholipid biosynthesis, as well as the development of the woundinduced respiration and cyanide resistance on aging, are restricted to thesuperficial 1 millimeter.

The respiration of fresh potato slices is three to five times thatof the intact tuber, and during approximately 24 h followingcutting it rises another 3- to 5-fold to yield the wound-inducedrespiration (15). Thus, the respiration of an aged slice may be 25times that of the intact tuber. A marked increase is observed withaging in the capacity for acetate incorporation into fatty acids(31), and choline incorporation into PL (5). Waring and Laties(30) proposed that the respiratory changes are a consequence ofmembrane biosynthesis.Whereas the respiration of aged slices is largely cyanide-insen-

sitive, and intact tuber respiration is stimulated by cyanide, freshslice respiration is predominantly cyanide sensitive (27). That thetricarboxylic acid cycle and glycolysis are inoperative in freshpotato slices has been deduced from their malonate resistance andtheir failure to evolve radioactive CO2 from '4C-labeled glucoseor from any labeled glycolytic or tricarboxylic acid cycle inter-mediates (14). With aging, malonate-resistant, cyanide-sensitivefresh slices become malonate-sensitive and cyanide-resistant (15,27). On the other hand tuber respiration is conventional, compris-ing glycolytic and tricarboxylic acid cycle metabolism as majorelements (15). Thus, fresh slice respiration differs from tuberrespiration.Jacobson et al. (12) have shown that tuber respiratory CO2 is

carbohydrate in origin, whereas fresh slice CO2 is predominantly

' This work was supported by grant GM 19807 from the United StatesPublic Health Service.

2Present address: Biological Sciences Department, Stanford University,Stanford, CA 94305.

3 Abbreviations: PL, phospholipid; FA, fatty acid; FFA, free fatty acid;TG, triglyceride; SE, sterol esters; PG, phosphatidyl-glycerol; PE, phos-phatidyl-ethanolamine; TGDG, trigalactosyl diglyceride; DGDG, digalac-tosyl diglyceride; MGDG, monogalactosyl diglyceride; CER, cerebroside;SG, sterol glucoside; ESG, esterified sterol glycoside; PI, phosphatidyl-inositol; SL, sulfolipid; UGL, unidentified glycoipid; CLAM, m-chloro-benzhydroxamic acid; GL, glycolipid; NL, neutral lipid; PC, phosphatidyl-choline; MG, monoglyceride; DG, diglyceride.

lipid in origin. Within a day the endogenous substrate of agedslices is once again carbohydrate. The evolution of CO2 fromlipids in fresh slices has been attributed in part to the a-oxidationof fatty acids on the basis of inhibition by imidazole, a ratherselective inhibitor of a-oxidation, and studies with carboxyl-la-beled long chain FA have reaffirmed the prevalence ofa-oxidationin fresh slices (16).

Ultrastructural studies (29) with beets have shown that in freshslices cellular membranes are grossly disorganized. Whereas lipidis sparse in potato tubers (10), the source of lipid for fresh slicerespiration is considered to be primarily cell membranes. Lipidacyl hydrolases as well as lipoxygenase are rife in potato (11).Herein, the quantitative changes in membrane lipids that accom-pany the cutting of intact potato tubers are reported, and supportthe proposition that membrane lipid degradation is the basis ofthe loss of CN resistance, with FFA arising from lipid breakdowninhibiting the conventional glycolytic and respiratory paths.The development of the induced respiration is inversely related

to slice thickness, whereas the immediate respiratory rise aftercutting is independent of thickness (15). The depth to whichcutting is sensed in relation to the immediate respiratory rise infresh slices is considerably greater than the depth through whichthe induced respiration subsequently develops with time (15).Accordingly, the spatial distribution of the induced and cyanide-resistant respiration has been examined in relation to lipid break-down and subsequent biosynthesis in an effort to determine theprimary factor(s) underlying the influence of slice thickness.

METHODS AND MATERIALS

PLANT MATERIAL

Potato tubers (Solanum tuberosum var. Russet Burbank) weregrown by the Department of Vegetable Crops, University ofCalifornia, Davis. Tubers were stored at 7 C and 90%o RH.

SLICE PREPARATION

Potato slices, 1 mm thick and 9 mm in diameter, were preparedand aged in conventional fashion (14, 27). In some instances agedslices were cut again into strips 2 mm wide.

RESPIRATORY MEASUREMENTS

Slice respiration was measured by conventional manometry at25 C. The estimation of '4CO2 evolution from carbon-labeledglucose was carried out as previously described (14).

LIPID METHODOLOGY

Sample Preparation.Intact Tuber. One mature potato tuber was used for each

experiment to avoid tissue heterogeneity. The tuber was washedthoroughly with distilled H20. Slices 1 mm x tuber diameter (20g fresh weight) were cut directly into boiling ethanol (96%) andboiled for 5 mi.

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THEOLOGIS AND LATIES

Slices. Fresh and aged slices 1 mm x 0.9 cm were prepared froma single tuber. Slices were boiled in nitrogen-saturated ethanol(96%) for 5 min previous to lipid extraction.

Lipid Extraction. The ethanolic extract was collected, and thelipids were extracted with chloroform:methanol (2:1, v/v) as de-scribed by Galliard (10). The crude lipid extract was washed with100 ml of 0.1 M NaCl, followed by three washings with 100 ml ofchloroform:methanol:0. 1 M NaCl (3:47:50) (2, 8). The lipid-con-taining chloroform phase was evaporated to dryness, and the lipidresidue, corresponding to 20 g fresh weight of tissue, was dissolvedin 20 ml benzene:ethanol (4:1, v/v). Aliquots of the lipid solutionwere taken for total lipid phosphorus assay (1), total lipid-hexose(21), and total FA analysis by GLC (18). The lipid solutions werestored under N2 at -20 C for further lipid fractionation.

Lipid Separation. A complete preparative separation for anal-ysis of each lipid was achieved by a combination of DEAE-cellulose (acetate form) column chromatography and TLC (17).DEAE-Cellulose Column Chromatography. The column proce-

dure developed by Rouser et al. (22), and used by Galliard forpotato lipid fractionation (10), was modified as follows: the potatolipids were fractionated into three main fractions containing neu-tral, zwitterionic, and acidic lipids, respectively, instead of the 10fractions prepared by Galliard. The three fractions were collectedunder 3 p.s.i. of nitrogen using the following solvents:

Frac- Solvent Lipids ElutedtionA 1. Chloroform Neutral (TG, SE, S)

B 2. Chloroform:methanol Zwitterionic and galacto-(3:2) + 0.2% acetic lipids (PG, PE, TGDG,acid DGDG, MGDG, FFA,

CER, SG, ESG)3. Methanol

C 4. Chloroform:methanol Acidic lipids (PI, DGDG,(2:1) + 10 g ammo- PG, SL, UGL)nium acetate/l + 20 mlNH4OH (28%)/l

Fraction C was treated by the method of Roughan and Batt (21)to remove ammonium acetate.

Thin-Layer Chromatography. Preparative TLC was performedon 500 ,um layers of silica gel G activated at 110 C for 60 min. Theindividual lipids of the DEAE fractions were separated with thefollowing developing solvents: (I) chloroform:methanol:aceticacid:water (170:30:20:5) (11). (II) Petroleum ether (30-60 C):dieth-ylether:acetic acid (70:30:1) (10). (III) Double development: (a)isopropyl ether:acetic acid (96:4), about 11 cm; (b) petroleumether:diethyl ether:acetic acid (90:10:1), about 17 cm. The individ-ual lipids of fractions B and C were separated by solvent system(I). The neutral lipids of fraction A were separated by solvents(II) or (III).

Lipid Identification. Lipid components were initially detectedin a separate narrow column on analytical TLC plates with iodinevapor with the following specific spray reagents (see [17]): (a) theVaskovsky and Kostetsky reagent for phospholipids; (b) the ben-zidine-metaperiodate reagent for GL; (c) the Dudzinski reagentfor FFA; (d) the phosphotungstic acid reagent and ferric chloridereagent for sterol-containing lipids. Additional confirmation ofindividual lipids was achieved by co-chromatography on TLCplates with standards of individual PL, TG, FA, and sterols.Gas Liquid Chromatography. FA analysis. Fatty acyl methyl

esters were prepared quantitatively according to the method ofMetcalfe et aL (18) from the initial lipid extracts, DEAE fractions,and individual lipids and FFA.The methyl esters were separated on an F and M Model 819-19

gas chromatograph equipped with a flame ionization detector anddual stainless steel columns (18.28 m x 0.63 cm) containing 15%HI-EFF-2BP ethylene glycol succinate on 80 to 100 mesh Chro-mosorb W (AW) (Applied Science Labs). The column was oper-ated isothermally at 180 C. The absolute amount of each FArelative to methyl palmitate was calculated from the area of thepeak divided by the relative detector response for each component.

Quantitative Analysis of Individual Lipids. The individual lipidswere separated by TLC, detected and quantitatively determinedby specific chemical tests for lipid-P (1), lipid galactose (21), or byGLC. The nonpolar lipids were eluted from the silica gel withpetroleum ether (30-60 C), while the polar lipids were quantita-tively extracted with chloroform:methanol:water:formic acid (97:97:4:2) (10).

BIOCHEMICALS

Linoleic acid, linolenic acid, and methyl palmitate were ob-tained from Sigma. BC13-methanol (12%, w/v) and Silica Gel G(Merck) were purchased from Supelco. Stainless steel GLC col-umns (18.28 m x 0.63 cm) packed with 15% HI-EFF-2BP ethyleneglycol succinate on 80/100 mesh Chromosorb W (AW), DEAEcellulose (exchange capacity 0.8-0.9 mEq/g dry weight) and astandard mixture ofFA methyl esters were obtained from AppliedScience Labs, Inc. CLAM was synthesized as previously described(26). 2',7'-Dichlorofluorescein, phosphotungstic acid, and 4-amino-3-hydroxyl-l-naphthalene sulfonic acid were purchasedfrom J. T. Baker Chemical Co. All organic solvents used were ofanalytical grade.

RESULTS

EFFECT OF POTATO TISSUE PROCESSING PRIOR TO LIPID EXTRACTIONON THE RECOVERY OF PL AND GL

During the early phase of this work, it was observed that thecontent of PL and GL varied, depending on the method ofprocessing the intact tissue prior to lipid extraction with chloro-form:methanol (2:1). Specifically, when potato tubers were sliceddirectly into liquid N2, and the frozen tissue subsequently boiledin 96% ethanol, PL was 392, and lipid galactose 408, ,umol/kgfresh weight, compared with 514 and 614 ,umol/kg fresh weightwhen tissue was cut directly into boiling alcohol-the decreasesbeing 24 and 34%, respectively. The lower polar lipid recoveryobserved with the first method was attributed to the activity ofphospho- and glycolipases as the frozen tissue momentarilyreached the optimum temperature for lipase activity. In order toassess accurately lipid levels in intact tissue, slices must be cutdirectly into boiling alcohol.Changes in PL and GL Content of Potato Tissues with Slicing

and Slice Aging. The quantitative changes in total lipid-phospho-rus and lipid-reducing sugar due to the slicing of an intact potatotuber, and subsequent slice aging, are shown in Table I. Lipidreducing sugar is predominantly galactose. The galactose levelsper se can be calculated from the table by first multiplying the di-and trigalactoside values by 2 and 3, respectively. Even when thesugar contribution of ESG is included there remains a 40%odisparity between total lipid reducing sugar as measured directly,and the sum of the galactose moieties as separately estimated. Thedisparity is attributed to unidentified GL, including the cerebro-sides, as well as to incomplete recovery of individual GL. Thetotal PL to GL ratio in an intact tuber is close to unity. Fresh 1-mm slices show a 35% loss in lipid-phosphorus and a 31% loss inlipid-reducing sugar after 2 h of incubation at 25 C. Slice agingleads to the resynthesis of the bulk of the degraded lipids. Furthercutting of an aged slice elicits no significant loss of membranelipids.

Fractionation of the total lipid extract from intact potato tuber

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WOUNDING AND MEMBRANE BREAKDOWN

Table I. The Effect of Slicing and Slice Aging on Potato LipidsFresh slices, 2 h from cutting at 25 C. Aged slices, 24 h at 25 C. Aged-

cut slices denote 24 h slices cut again in 2 mm transverse strips andincubated for I h.

Intact Fresh Aged Aged-Lipid Components Tuber Slices Slices Cut

Slices

,umol/kgfresh wt

PhospholipidsTotal lipid P 660 433 670 628Zwitterionic lipid P (fraction B) 570 380 540 480Acid lipid P (fraction C) 150 115 160 150PC 311 183 322 289PE 205 133 188 179PI 57 43 66 55PG 14 24 21DPDG 6 15 12

GlycolipidsTotal lipid reducing sugar 709 486 684 672MGDG 73 43 80 80DGDG 84 48 90 80TGDG 28 22 30 27SL 10 7 10 10ESG 72 67 47 56

Neutral lipidsTG 144 63 54 60

Free fatty acids 75 124 130 139

by DEAE-cellulose column chromatography distributes total lipidphosphorus unequally in two fractions. Fraction B, which containsthe zwitterionic PL, represents 70 to 80%1o ofpotato tuber PL, whilethe remaining 25% comprises the acidic PL (fraction C). FractionA contains the phosphorus-free NL. When a potato tuber is slicedthe predominant zwitterionic PL decrease by a third, while theacidic PL drop 24%. Slice aging leads to an increase in both classesof PL. The cutting of an aged slice has no effect on lipid content.Changes in Individual Lipids after Slicing. Table I further

defines the quantitative changes of membrane lipids in severalclasses after slicing and subsequent slice aging. The lipids of anintact potato tuber are a complex mixture of PL, galactolipids,and sterol-containing GL (see [10]), the essential components ofpotato membranes. On the other hand, TG constitute only 17% ofthe total potato lipids. PC and PE are the most abundant PL,while MGDG and DGDG are the predominant galactolipids.Among the sterol-containing lipids, ESG is the main constituent.SG and free sterols are minor components. Potato tissue is low inFFA. The FFA fraction is a mixture of 14:0, 16:0, 18:0, and 18:1.Free polyunsaturated fatty acids such as linoleic (18:2) and lino-lenic (18:3) are scarce in potato, constituting 5 to 10%1o of the totalFFA fraction (data not shown).PC and PE decrease 41 and 35%, respectively, with slicing. The

acidic PL, PI, decreases 24%. Among the galactolipids, theMGDGand DGDG are the most susceptible to destruction. ESG, themain sterol lipid, undergoes minor change. The TG decrease morethan 55%, while there is a doubling in the FFA content in freshslices. Slice FFA is enriched with polyunsaturated FA. Whereas18:2 and 18:3 are minor components (7%) of tuber FFA, theyrepresent about 22% ofFFA in fresh slices. Although the increasein FFA in fresh slices reflects the burst of lipid acyl hydrolaseactivity that attends slicing, measurable FFA does not account forall the PL and GL which disappears. A large part of the disparityis probably due to FA hydroperoxide formation, there being a

considerable quantity of the latter retained near the origin follow-

ing TLC separation of the neutral lipid fraction. Table I furthershows that slice aging results in a net synthesis of PL and GL. PCand MGDG and DGDG increase most extensively. The FFAfraction remains quantitatively the same, but the percentage of 18:2 and 18:3 drops from 22 to 12% in this fraction (data not shown).Further cutting of aged slices causes no significant change in thePL or GL levels, in sharp contrast to the effect of slicing an intacttuber.

Fatty Acid Content and Composition of Potato Lipids.FA of the Total Lipid Extract. The quantitative changes of the

total esterified FA in various potato tissues are shown in Table II.Potato tuber lipid comprises five main FA with the order ofabundance 18:2 > 18:3 >16:0 > 18:0 > 18:1. The two polyunsat-urated FA 18:2 and 18:3 constitute 75% of the total FA. Slicingresults in a 35 and 57% decrease of 18:2 and 18:3, respectively,while the saturated FA, palmitate and stearate, drop 25 and 20%,respectively. Thus, the loss oflipid-phosphorus and lipid-galactosein fresh tissue is due neither to phospholipase C nor galactosidaseactivity, but seemingly to a surge of lipid acyl hydrolase activity.

Table II. The Effect of Slicing and Slice Aging on Potato Fatty AcidsDesignations as per Table I.

Intact Fresh Aged Aged-Lipid Components Tuber Slices Slices Cut

Slices

l.tmol/kgfresh wt

Total lipidsTotal fatty acids

16:016:117:018:018:118:218:3

2,610500

Trace0

10050

1,360600

Neutral lipids (fraction A)Total fatty acids

16:016:117:018:018:118:218:3

Glycolipids and zwitterionic lip-ids (fraction B)

Total fatty acids16:016:117:018:018:118:218:3

Acid lipids (fraction C)Total fatty acids

16:016:117:018:018;118:218:3

43384200

1812

177122

1,786341

Trace0

8136

967360

28287

Trace0

126

111

66

1,690400

Trace308040900240

18932332284

6526

1,146256

Trace05436

591208

21858

Trace0116

8852

2,710580700

110140

1,280530

162481404106125

1,812350

Trace0

9181

922368

392105

Trace0

232717166

2,730590800

105120

1,320515

181461608107030

1,737345

Trace0

7381

863375

34890

Trace0182115663

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THEOLOGIS AND LATIES

This conclusion is fortified by the absence of diglycerides in freshslices. The appearance of an odd chain FA such as 17:0 in freshslices is of significance, since it suggests a-oxidase activity (16).Slice aging results in a net synthesis of linoleic, linolenic, andpalmitic acids (31). Moreover, the appearance of palmitoleic acid(16:1), and the increase of oleic (18:1) acid to a level higher thanthat of stearic acid, constitute a characteristic change in the FAcomposition induced by slice aging. Whereas cutting an intacttuber results in an extensive loss in FA, cutting an aged disk hasno effect.

Fatty acids of Lipid Fractionsfrom DEAE Column Chromatog-raphy. The content of individual FA ofthe principal lipid fractionsfrom various potato tissues is also set out in Table II. Fraction Acontains 17% of the total FA in potato tuber. TG are the mainNL, while SE, MG, DG, and free sterols constitute minor com-

ponents. Linoleic and linolenic acids comprise 69% of the FA inthis fraction, which is decreased 56% by slicing. The loss oflinolenic acid is as great as 80%o. Thus, the level of the twopolyunsaturated FA in the NL drops from 69 to 41% after slicing.Slice aging induces no quantitative changes in this fraction. Sev-enty per cent of the total FA in potato tuber are recovered infraction B. Linoleic and linolenic acids represent 74% of the totalFA, while palmitate is the main saturated acid (19%). Slicing leadsto a 35% decrease of FA in this fraction. The two polyunsaturatedFA drop as much as 40o, while palmitate undergoes a smaller

change (25%). The FA content increases during slice aging. Oleicacid (18:1) undergoes a dramatic increase (280%) compared withthe intact tuber level. The FA of fraction C represent 11% of thetotal FA in potato tuber. Although slicing results in the lowestpercentage drop in total FA in fraction C (22%), acids of thisfraction undergo the largest proportional changes during sliceaging. For example, linoleic acid increases 54% above the intacttuber level, while oleic acid undergoes a 450% increase.

CONTROLLING INFLUENCE OF THICKNESS ON MEMBRANE LIPIDBREAKDOWN, BIOSYNTHESIS AND DEVELOPMENT OF THE CYANIDE-

RESISTANT RESPIRATION IN POTATO SLICES

Early respiratory studies of potato slices showed the develop-ment of wound-induced respiration to be inversely related tothickness (see [11]). Whereas respiratory development in thin slicesis accompanied by a spate of PL degradation (Table I), anddepends upon subsequent PL biosynthesis (30), a study was un-dertaken on the effect of slice thickness on lipid metabolism, todetermine whether the absence of induced respiration in thickslices reflects a lack of PL synthesis therein.When a core of tissue 3 cm in diameter and 5 cm deep is cut

from within the vascular ring of the tuber, PL drops 10%1o in 5 min,and 21% in 15 min at 3 C. The corresponding loss in total FA is18 and 38%, respectively. The drop extends throughout the tissueas can be demonstrated by comparing the lipid content of aninternal 2 cm cylinder with that of a peripheral ring of tissue 1 cmthick (data not shown). Thus, the degradative signal travels rapidlyto a considerable depth even at low temperatures, and lipiddegradation per se has a low temperature coefficient.The external appearance of an aged thick potato slice or core

(20 mm thick x tuber diameter) resembles an aged 1 mm slice.Both develop a pink-tan color, but the specific respiratory rate ofthe thin slice is much greater. The surface millimeter of an agedthick slice has a respiratory rate approaching that of a 1-mm thickaged disc, whereas slices obtained from deep-seated tissue resem-ble fresh thin (1 mm) slices (Table III).. The development of thewound-induced respiration is restricted to the core surface.When a thick potato core (2 cm thick x diameter of tuber) is

aged for 24 h the surface millimeter develops the wound-inducedrespiration and alternative path, much as does a 1-mm aged slice(Table III). A slice 10 mm from the core face resembles theordinary conventional fresh thin potato slice, i.e., its respiration is

Table III. Effect of Tissue Depth on the Development of the Wound-Induced and CN-Resistant Respiration and on Phospholipid and Fatty Acid

Content in Slicesfrom Potato CoresAging carried out for 36 h.

Aged CoresSlices (1 mm) (2 mm x Tuber

Diameter)

Inhibitor 10th1 mm mm

Fresh Aged Core fromface Core

face

Al 02/gfresh wt.hControl 43 167 121 40KCN,0.lmM 11 97 60 14CLAM, 2 mM 39 117 116 41KCN + CLAM 10 30 26 14Antimycin, 10,UM 13 188 118 21Antimycin + CLAM 7 83 62 15CCCP,a 10 JiM 98 172 164 63

,umol/kgfresh wt

Lipid-P 412 560 515 362Total FA 1,960 2,300 2,100 1,50016:1 Trace 113 101 Trace18:1 57 160 160 2018:2 1,010 1,035 845 84418:3 352 385 427 260

a CCCP, carbonyl-cyanide m-chlorophenyl hydrazone.

Table IV. Effect of Tissue Depth on the Development of CN-ResistantRespiration and Utilization of Glucose and Citrate by Potato Slices Taken

from Aged CoresSlices I mm thick. Slice diameter, 0.9 cm; core, 2 cm thick, diameter of

tuber. Tissue aged 48 h where indicated. Initial radioactivity ofthe externalsolution: [U-'4Cjglucose, 18.6 x 106 dpm/15 ml. [1,5 14CJcitrate, 21.2 x 106dpm/15 ml.

Respiration 14CO2 Release Uptake

Tissue Co-KCN, l- Cttrol

0.1 Glucose Citrate cou- Cit-trol cose ratemM

jIl 02/gfresh dpm x 10-4/3 g % initialwt h fresh wt. 2 h radioactivity

taken up

SlicesFresh 40 12 0.24 0 6 2Aged 121 93 120 223 95 61

Aged coresFirst mm, core

face 117 80 41 103 62 24Second mm from

face 46 18 14 0.7 19 6Third mm from

face 42 10 3 0 10 2Fourth mm from

face 35 15 2 0 8 1

sensitive to cyanide and antimycin, and it shows no alternativepath activity.The 1st mm from the core face has a FA content similar to the

1 mm aged slice, whereas the deep seated tissue (10th mm) fails tosynthesize new FA, and its FA content is lower than that of freshly

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PPol1WOUNDING AND MEMBRANE BREAKDOWN

cut 1 mm discs, due to continuous lipid destruction. The increaseofpalmitoleic (16:1) and oleic (18:1) acids observed in the coreface strongly indicates that the surface tissue undergoes the bio-chemical alterations of a thin slice.Comparison of Substrate Utilization by Superficial and Deep-

Seated Potato Tissue. Table IV compares the rate Of "'CO2evolution from "C-labeled glucose and citrate by thin fresh andaged slices as well as from deep-seated potato tissue. The aging ofthin potato slices enhances exogenous substrate utilization as haspreviously been shown (14). The enhancement of glucose andcitrate utilization with aging in thick potato slices is confined tothe surface tissue. In this characteristic too, aged deep-seated tissueresembles fresh slices.

DISCUSSION

The preparation of fresh slices from potato tubers alters therespiratory metabolism from carbohydrate to lipid. Evidence pre-sented herein suggests that the FA serving fresh slice respirationarise from the breakdown of cellular membranes. The extensiveloss in lipid-phosphorus, GL, and polyunsaturated FA observedin freshslices is attributed to a combined action oflipid acylhydrolases, lipoxygenase, and FA hydroperoxide degradative en-zymesall of which are plentiful in potato(11). Slicing appears toinitiate lipid peroxidation, as indicated by the preferential decreasein polyunsaturated linoleic and linolenic acids. It has recentlybeen shown (13) that fresh potato slices produce ethane, a sensitivemarker of lipid peroxidation. Looking to TableII, the GL/zwit-terionic and neutral fractions show the greatest fractional drop intotal FA in response toslicing. On an absolute basis, however, thezwitterionic fraction accounts for the largest loss. The FFA re-leased may be markedly inhibitory to electron transport andoxidative phosphorylation (32), as well as to glycolysis (19) andglucose transport (6). Accordingly, inhibition of conventionalmetabolism (glycolysis and the tricarboxylic acid cycle) in freshpotatoslices has been attributed by us to the generation of FFAon slicing (28). The suppression of phosphorylation by FFA inturn has been ascribed to the inhibition of the mitochondrialADP/ATP antiport (32). Parenthetically, we have proposed (28)that the marked stimulation of fresh slice respiration by uncoupleris due to the release of this inhibition, in analogy to the effect ofuncoupler on oligomycin treated mitochondria.Mass spectrophotometric analysis of respiratory CO2 from fresh

potato slices indicates that70%o of the total CO2 is lipid in origin(12)-a larger fraction of lipid carbon than is accounted for bya-oxidation (16). Although,8-oxidation takes place in freshslices,the acetyl product is not oxidized in a conventional way. Citrateis heavily labeled when ['4C]palmitate-l- or [2-'4Clacetate (14) isgiven as exogenous substrate in fresh slices, and it remains to beseen whether fresh potato slices utilize acetyl-CoA in ketone bodyformation (acetoacetate, f-hydroxybutyrate and acetone) and ac-cumulation. Ketone bodies may supply a part of the respiratoryCO2 in fresh tissue, inasmuch as it was found that exogenous [1-"4Cjpropane- 1 ,2-diol, an intermediate of acetoacetate metabolism,is readily utilized by fresh potato slices (26).

Controlling Influence of Thickness on Metabolism. The biosyn-thetic events related to membrane biosynthesis are suppressed indeep-seated tissue (Table IV). In addition to PL and membranebiosynthesis (30) and protein and RNA synthesis (24), DNA andsuberin biosynthesis with slice aging are also confined to thesurface (3), as is the appearance of isoperoxidases (4). In short, thebasic events which lead to the development of the induced respi-ration as well as to CN-resistance in potato slices are limited tothe surface. The extent to which 02 tension controls developmenthas been discussed extensively (15).

Cyanide Resistance in Relation to Membrane Integrity. Theslicing of potato tubers not only alters the respiratory metabolismfrom carbohydrate to lipid, but also produces CN-sensitive fresh

slices from the CN-resistant parent organ. Lipid breakdown mayentail the degradation of one or more mitochondrial membranecomponents essential to the operation of the CN-resistant path (cf.[9]); or products of lipid breakdown (FFA, lysolecithin, FA hy-droperoxides) may have an inhibitory effect on the alternativeoxidase (see [7]). Lipid degradation due to slicing has widespreadeffects. For example, Rungie and Wiskich (23) have shown thatNADH-Cyt c reductase activity is absent in microsomes fromfresh turnip slices while present in microsomes from the intactorgan. Fresh turnip slices suffer extensive lipid breakdown (28).Furthermore, slicing an intact storage organ initiates not onlymembrane lipolysis, but also the destruction of other structuralcell components such as ribosomal RNA (25). The developmentof microsomal P-450 mediated hydroxylations in aged slices (20)raises the prospect that microsomal enzymes too may be disruptedon slcing.Our presumption that CN insensitivity is correlated with mem-

brane integrity has been strengthened by the observation that agedslices show neither lipid breakdown nor loss of CN resistance onfurther cutting, and the correlation between the absence of lipidbreakdown and the persistence of CN resistance in fresh slices ofa variety of fleshy storage organs is good (28). The absence oflipid breakdown in aged slices on cutting is not due to the absenceof lipolytic enzymes since lipid acyl hydrolase activity is demon-strable to the same degree in homogenates of fresh and aged slices.The explanation must be sought in terms of membrane confor-mation or compartmentation. Whether membrane lipid break-down in freshslices caused by cutting is due to an activation oflipolytic enzymes, or, for whatever reason, an enhanced suscepti-bility of membranelipid to lipid acyl hydrolase attack, a fullexplanation of the phenomenon must take into account the rapid-ity, the effective distance, and the relative temperature insensitivityof the signal for lipid degradation.The development of the wound-induced respiration in potato

slices represents a complex phenomenon comprising the releasefrom inhibition of glycolysis and the tricarboxylic acid cyclestemming from the oxidative scavenging of FFA, and a spate oflipid biosynthesis related to membrane formation. Whereas theonset and course of the induced respiration depends on RNA andprotein synthesis (24), de novo synthesis of mitochondria is notinvolved (28). By contrast, PL biosynthesis is indispensable (30).Wounding causes membrane-lipid breakdown in conjunction withanomalous respiration, while recovery from wounding entailsmembranelipid biosynthesis with the reestablishment and aug-mentation of normal respiration.

Acknowledgments-A. Theologis is indebted to UCLA for a predoctoral fellowshipand to Phi Beta Kappa for a foreign student predoctoral scholarship. Our thanks aredue Professor Herman Timm, Department of Vegetable Crops, University of Cali-fornia, Davis, for the potato tubers used in this study.

LITERATURE CITED

1. BARTLETT GR 1959 Phosphorus assay in column chromatography. J Biol Chem234: 466-468

2. BLIGH EG, WJ DYER 1959 A rapid method of totallipid extraction andpurification. Can J Biochem Physiol 37: 911-917

3. BORCHERT R, JD MCCHESNEY 1973 Time course and localization of DNAsynthesis during wound healing of potato tuber tissue. Dev Biol 35: 293-301

4. BORCHERT R 1974 Isoperoxidases as markers of the wound-induced differentia-tion pattern of potato tuber. Dev Biol 36: 391-399

5. CASTELFRANCO PA, WJ TANG, ML BOLAR 1971 Membrane transformation inaging potato tuber slices. Plant Physiol 48: 795-800

6. DECKER M, W TANNER 1975 Rapid release of free fatty acids during cell breakageand their effect on a sugar-proton cotransport system in Chlorella vulgaris. FedEur Biochem Soc Lett60: 346-348

7. DOBRETsov GE, TA BORSCHEVSKAYA, VA PETROV, YuA VLADIMIROV 1977 The

increase of phospholipid bilayer rigidity after lipid peroxidation. FEBS Lett84: 125-128

8. FOLCH J, M LEES, GHS STANLEY 1957 A simple method for the isolation andpurification of total lpids from animal tissues. J Biol Chem 226: 497-509

9. FOURCANs B 1974 Role of phospholipids in transport and enzymic reactions. AdvLipid Res 12: 148-226

57Plant Physiol. Vol. 68, 1981

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58 THEOLOGIS

10. GALLIARD T 1968 Aspects of lipid metabolism in higher plants. L. Identificationand quantitative determination of the lipids in potato tubers. Phytochemistry7:1907-1914

11. GALLIARD T 1970 The enzymic breakdown of lipids in potato tuber by phospho-lipid- and galactolipid-acyl hydrolase activities and by lipoxygenase. Phyto-chemistry 9: 1725-1734

12. JACOBSON BS, BN SMiTH, S EPSTEIN, GG LATIES 1970 The prevalence ofcarbon-13 in respiratory carbon dioxide as an indicator of the type of endogenoussubstrate. The change from lipid to carbohydrate during the respiratory rise inpotato slices. J Gen Physiol 55: 1-17

13. KONZE JR, EF ELsTNmE 1978 Ethane and ethylene formation by mitochondriaas indication of aerobic lipid degradation in response to wounding of planttissue. Biochim Biophys Acta 528: 213-221

14. LAnEs GG 1964 The onset of tricarboxylic acid cycle activity with aging inpotato slices. Plant Physiol 39: 654-663

15. LATIES GG 1978 The development and control of respiratory pathways in slicesof plant storage organs. In G Kahl, ed, Biochemistry of Wounded PlantTissues. W de Gruyter, Berlin, New York, pp 421-466

16. LATES GG, C HoE-ai±, BS JACOBSON 1972 a-Oxidation of endogenous fattyacids in fresh potato slices. Phytochemistry 11: 3403-3411

17. MARINETn, GV 1969 Lipid chromatographic analysis, vol 2. Marcel Dekker,New York

18. METCALFE, LD, AA SMITH, JR PELxA 1966 Rapid preparation of fatty acid estersfrom lipids for gas chromatographic analysis. Anal Chem 38: 514-515

19. RAMADOss CS, K UYEDA, JM JOHNSTON 1976 Studies on the fatty acid inacti-vation of phosphofructokinase. J Biol Chem 251: 98-107

20. RICH PR, CJ LAMB 1977 Biophysical and enzymological studies upon theinteraction of trans-cinnamic acid with higher plant microsomal cytochromesP.450. Eur J Biochem 72: 353-360

21. ROUGHAN PG, RD BArr 1968 Quantitative analysis of sulfolipid (sulfoquino-vosyl diglyceride) and galactolipids (monogalactosyl and digalactosyl diglyc-erides) in plant tissues. Anal Biochem 22: 74-88

kND LATIES Plant Physiol. Vol. 68, 1981

22. RouSER G, G KRITCHEVSKY, A YAMAMOTO, G SIMON, C GALLI, AJ BAUMAN1969 Diethylaminoethyl and triethylaminoethyl cellulose column chromato-graphic procedures for phospholipids, glycolipids and pigments. MethodsEnzymol 14: 272

23. RUNGIE JM, JT WISKICH 1972 Changes in microsomal electron transport ofplant storage tissues induced by slicing and aging. Aust J Biol Sci 25: 103-113

24. SAMPSON MJ, GG LATIES 1968 Ribosomal RNA synthesis in newly sliced discsof potato tuber. Plant Physiol 43: 1011-1016

25. SPARKUHL J, RL GARE, G SEarRFIELD 1976 Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices. Planta 129:97-104

26. THEOLOGIs A 1979 The genesis, development and participation of cyanideresistant respiration in plant tissues. PhD thesis. University of California, LosAngeles

27. THEOLOGIs A, GG LATIES 1978 Relative contribution of cytochrome-mediatedand cyanide-resistant electron transport in fresh and aged potato slices. PlantPhysiol 62: 232-237

28. THEOLOGIS A, GG LATIES 1980 Membrane-lipid breakdown in relation to thewound-induced and cyanide resistant respiration in tissue slices. A comparativestudy. Plant Physiol 66: 890-896

29. VAN STEVENINCK RFM, ME JACKMAN 1967 Respiratory activity and morphologyof mitochondria isolated from whole and sliced storage tissue. Aust J Biol Sci20: 749-760

30. WARING AJ, GG LATIES 1977 Inhibition of the development of the inducedrespiration and cyanide insensitive respiration in potato tuber slices by ceru-lenin and dimethylaminoethanol. Plant Physiol 60: 11-16

3 1. WILLEMOT G, PK STUMPF 1967 Fat metabolism in higher plants. XXXIV.Development of fatty acid synthetase as a function of protein synthesis in agingpotato tuber slices. Plant Physiol 42: 391-397

32. WOJTCZAK L, K BOGUCKA, MG SARZALA, H ZALuSKA 1969 Effect of fatty acidson energy metabolism and the transport ofadenine nucleotides in mitochondriaand other cellular structures. FEBS Symp 17: 79-92

A

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